研究者データベース

FOX BRIAN GRANT(フオツクス ブライアン グラント)
農学研究院 食水土資源グローバルセンター
教授

基本情報

所属

  • 農学研究院 食水土資源グローバルセンター

職名

  • 教授

J-Global ID

研究分野

  • ライフサイエンス / 構造生物化学

研究活動情報

論文

  • Keisuke Ohashi, Shogo Hataya, Akane Nakata, Kazuki Matsumoto, Natsumi Kato, Wakana Sato, Camila Carlos-Shanley, Emily T Beebe, Cameron R Currie, Brian G Fox, Taichi E Takasuka
    Applied and environmental microbiology 2021年05月14日 
    The cellulolytic insect symbiont bacterium, Streptomyces sp. SirexAA-E (SirexAA-E) secretes a suite of Carbohydrate Active enZymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by LC-MS/MS. The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. By genomic analysis, we found a unique 12 bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and so are likely under the regulation of both SsManR and SsCebR.ImportanceStreptomyces sp. SirexAA-E, a microbial symbiont of biomass harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.
  • Evan M Glasgow, Elias I Kemna, Craig A Bingman, Nicole L Ing, Kai Deng, Christopher M Bianchetti, Taichi E Takasuka, Trent R Northen, Brian G Fox
    The Journal of biological chemistry 2020年10月16日 
    Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of ten new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and mass spectrometry. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.
  • Glasgow EM, Vander Meulen KA, Takasuka TE, Bianchetti CM, Bergeman LF, Deutsch S, Fox BG
    Journal of molecular biology 431 6 1217 - 1233 2019年03月 [査読有り][通常論文]
  • Kontur WS, Bingman CA, Olmsted CN, Wassarman DR, Ulbrich A, Gall DL, Smith RW, Yusko LM, Fox BG, Noguera DR, Coon JJ, Donohue TJ
    The Journal of biological chemistry 293 14 4955 - 4968 2018年04月 [査読有り][通常論文]
  • Ryan P. McAndrew, Noppadon Sathitsuksanoh, Michael M. Mbughuni, Richard A. Heins, Jose H. Pereira, Anthe George, Kenneth L. Sale, Brian G. Fox, Blake A. Simmons, Paul D. Adams
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 30 E6029 - E6030 2017年07月 [査読有り][通常論文]
  • Benjamin B. Minkoff, Shin-ichi Makino, Miyoshi Haruta, Emily T. Beebe, Russell L. Wrobel, Brian G. Fox, Michael R. Sussman
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 14 5932 - 5942 2017年04月 [査読有り][通常論文]
     
    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.
  • Justin F. Acheson, Lucas J. Bailey, Thomas C. Brunold, Brian G. Fox
    NATURE 544 7649 191 - + 2017年04月 [査読有り][通常論文]
     
    Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O-2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe2+/Fe3+ species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.
  • Johnnie A. Walker, Sivakumar Pattathil, Lai F. Bergeman, Emily T. Beebe, Kai Deng, Maryam Mirzai, Trent R. Northen, Michael G. Hahn, Brian G. Fox
    BIOTECHNOLOGY FOR BIOFUELS 10 31  2017年02月 [査読有り][通常論文]
     
    Background: Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. Results: Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose-and xylose-backbone polysaccharides. Conclusions: The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oximeNIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.
  • Ryan P. McAndrew, Noppadon Sathitsuksanoh, Michael M. Mbughuni, Richard A. Heins, Jose H. Pereira, Anthe George, Kenneth L. Sale, Brian G. Fox, Blake A. Simmons, Paul D. Adams
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 50 14324 - 14329 2016年12月 [査読有り][通常論文]
     
    Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 angstrom), its product vanillin (1.75 angstrom), and without any bound ligand (1.61 angstrom). The enzyme is a seven-bladed beta-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.
  • Bo Li, Shin-ichi Makino, Emily T. Beebe, Daisuke Urano, David J. Aceti, Tina M. Misenheimer, Jonathan Peters, Brian G. Fox, Alan M. Jones
    PROTEIN EXPRESSION AND PURIFICATION 126 33 - 41 2016年10月 [査読有り][通常論文]
     
    Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on G alpha (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on G alpha, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on G alpha and can be purified to a level suitable for biochemical analyses. (C) 2016 Elsevier Inc. All rights reserved.
  • Adam J. Book, Gina R. Lewin, Bradon R. McDonald, Taichi E. Takasuka, Evelyn Wendt-Pienkowski, Drew T. Doering, Steven Suh, Kenneth F. Raffa, Brian G. Fox, Cameron R. Currie
    PLOS BIOLOGY 14 6 2016年06月 [査読有り][通常論文]
     
    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.
  • Book AJ, Lewin GR, McDonald BR, Takasuka TE, Wendt-Pienkowski E, Doering DT, Suh S, Raffa KF, Fox BG, Currie CR
    PLoS biology 14 6 e1002475  2016年06月 [査読有り][通常論文]
  • Gina R. Lewin, Camila Carlos, Marc G. Chevrette, Heidi A. Horn, Bradon R. McDonald, Robert J. Stankey, Brian G. Fox, Cameron R. Currie
    ANNUAL REVIEW OF MICROBIOLOGY, VOL 70 70 235 - + 2016年 [査読有り][通常論文]
     
    The ancient phylum Actinobacteria is composed of phylogenetically and physiologically diverse bacteria that help Earth's ecosystems function. As free-living organisms and symbionts of herbivorous animals, Actinobacteria contribute to the global carbon cycle through the breakdown of plant biomass. In addition, they mediate community dynamics as producers of small molecules with diverse biological activities. Together, the evolution of high cellulolytic ability and diverse chemistry, shaped by their ecological roles in nature, make Actinobacteria a promising group for the bioenergy industry. Specifically, their enzymes can contribute to industrial-scale breakdown of cellulosic plant biomass into simple sugars that can then be converted into biofuels. Furthermore, harnessing their ability to biosynthesize a range of small molecules has potential for the production of specialty biofuels.
  • Johnnie A. Walker, Taichi E. Takasuka, Kai Deng, Christopher M. Bianchetti, Hannah S. Udell, Ben M. Prom, Hyunkee Kim, Paul D. Adams, Trent R. Northen, Brian G. Fox
    BIOTECHNOLOGY FOR BIOFUELS 8 220  2015年12月 [査読有り][通常論文]
     
    Background: Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. Results: CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. Conclusion: We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.
  • Justin F. Acheson, Hannah Moseson, Brian G. Fox
    BIOCHEMISTRY 54 38 5980 - 5988 2015年09月 [査読有り][通常論文]
     
    The 1.6 angstrom crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron-sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface.
  • Yonghong Bai, Jason G. McCoy, Elena J. Levin, Pablo Sobrado, Kanagalaghatta R. Rajashankar, Brian G. Fox, Ming Zhou
    NATURE 524 7564 252 - + 2015年08月 [査読有り][通常論文]
     
    Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs1-4. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity(5,6). Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases(7). SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl-or palmitoyl-CoA(8,9). The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b(5), which regenerates the di-iron centre(10). To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 angstrom resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.
  • Aceti DJ, Bingman CA, Wrobel RL, Frederick RO, Makino S, Nichols KW, Sahu SC, Bergeman LF, Blommel PG, Cornilescu CC, Gromek KA, Seder KD, Hwang S, Primm JG, Sabat G, Vojtik FC, Volkman BF, Zolnai Z, Phillips GN Jr, Markley JL, Fox BG
    Journal of structural and functional genomics 16 2 67 - 80 2015年06月 [査読有り][通常論文]
  • Christopher M. Bianchetti, Taichi E. Takasuka, Sam Deutsch, Hannah S. Udell, Eric J. Yik, Lai F. Bergeman, Brian G. Fox
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 19 11819 - 11832 2015年05月 [査読有り][通常論文]
     
    Background: SacteLam55A is a GH55 enzyme from highly cellulolytic Streptomyces sp. SirexAA-E. Results: Substrate-bound structures identify residues involved in binding, catalysis, enforcement of reaction specificity, and possibly processivity. Conclusion: Natural GH55 are exo--1,3-glucanases with a broad range of temperature and pH optima. Significance: Experimental annotation of GH phylogenetic space by use of bioinformatics, high throughput cell-free translation, biochemical assay, and structure determination is feasible. The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo--1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 -1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on approximate to 30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.
  • Elizabeth J. Blaesi, Brian G. Fox, Thomas C. Brunold
    BIOCHEMISTRY 54 18 2874 - 2884 2015年05月 [査読有り][通常論文]
     
    Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-cysteine (Cys) to cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation similar to 100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 -> Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe(II)CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe(II)CDO possesses a six-coordinate Fe(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the cysteine-tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe(II)CDO [which mimics the (O-2/Cys)-CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe(II)CDO active site.
  • Deng K, Guenther JM, Gao J, Bowen BP, Tran H, Reyes-Ortiz V, Cheng X, Sathitsuksanoh N, Heins R, Takasuka TE, Bergeman LF, Geertz-Hansen H, Deutsch S, Loqué D, Sale KL, Simmons BA, Adams PD, Singh AK, Fox BG, Northen TR
    Frontiers in bioengineering and biotechnology 3 153  2015年 [査読有り][通常論文]
  • Deng K, Takasuka TE, Bianchetti CM, Bergeman LF, Adams PD, Northen TR, Fox BG
    Frontiers in bioengineering and biotechnology 3 165  2015年 [査読有り][通常論文]
  • Elizabeth J. Blaesi, Brian G. Fox, Thomas C. Brunold
    BIOCHEMISTRY 53 36 5759 - 5770 2014年09月 [査読有り][通常論文]
     
    Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron-dependent enzyme that converts exogenous cysteine (Cys) to cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe(III)CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe(III), but not the Fe(II) center. An analogous analysis carried out for Cys-Fe(III)CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe(III) center to generate a low-spin (S = 1/2) Fe(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 angstrom of the cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys.
  • Justin F. Acheson, Lucas J. Bailey, Nathaniel L. Elsen, Brian G. Fox
    NATURE COMMUNICATIONS 5 5009  2014年09月 [査読有り][通常論文]
     
    Productive biomolecular recognition requires exquisite control of affinity and specificity. Accordingly, nature has devised many strategies to achieve proper binding interactions. Bacterial multicomponent monooxygenases provide a fascinating example, where a diiron hydroxylase must reversibly interact with both ferredoxin and catalytic effector in order to achieve electron transfer and O-2 activation during catalysis. Because these two accessory proteins have distinct structures, and because the hydroxylase-effector complex covers the entire surface closest to the hydroxylase diiron centre, how ferredoxin binds to the hydroxylase has been unclear. Here we present high-resolution structures of toluene 4-monooxygenase hydroxylase complexed with its electron transfer ferredoxin and compare them with the hydroxylase-effector structure. These structures reveal that ferredoxin or effector protein binding produce different arrangements of conserved residues and customized interfaces on the hydroxylase in order to achieve different aspects of catalysis.
  • Ming Yu, Yingcai Wang, Jiang Zhu, Michael D. Bartberger, Jude Canon, Ada Chen, David Chow, John Eksterowicz, Brian Fox, Jiasheng Fu, Michael Gribble, Xin Huang, Zhihong Li, Jiwen (Jim) Liu, Mei-chu Lo, Dustin McMinn, Jonathan D. Oliner, Tao Osgood, Yosup Rew, Anne Y. Saiki, Paul Shaffer, Xuelei Yan, Qiuping Ye, Dongyin Yu, Xiaoning Zhao, Jing Zhou, Steven H. Olson, Julio C. Medina, Daqing Sun
    ACS MEDICINAL CHEMISTRY LETTERS 5 8 894 - 899 2014年08月 [査読有り][通常論文]
     
    Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.
  • Adam J. Book, Gina R. Lewin, Bradon R. McDonald, Taichi E. Takasuka, Drew T. Doering, Aaron S. Adams, Joshua A. V. Blodgett, Jon Clardy, Kenneth F. Raffa, Brian G. Fox, Cameron R. Currie
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 80 15 4692 - 4701 2014年08月 [査読有り][通常論文]
     
    Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.
  • Jo E. Lomax, Christopher M. Bianchettl, Aram Chang, George N. Phillips, Brian G. Fox, Ronald T. Raines
    JOURNAL OF MOLECULAR BIOLOGY 426 17 3041 - 3056 2014年08月 [査読有り][通常論文]
     
    Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI center dot RNase complexes to a resolution of 2.20, 2.21, and 1.92 angstrom, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in Rls from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology. (C) 2014 Published by Elsevier Ltd.
  • Adam J. Book, Ragothaman M. Yennamalli, Taichi E. Takasuka, Cameron R. Currie, George N. Phillips, Brian G. Fox
    BIOTECHNOLOGY FOR BIOFUELS 7 109  2014年08月 [査読有り][通常論文]
     
    Background: Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families. Results: Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site. Conclusions: The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.
  • Taichi E. Takasuka, Christopher M. Bianchetti, Yuki Tobimatsu, Lai F. Bergeman, John Ralph, Brian G. Fox
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 82 7 1245 - 1257 2014年07月 [査読有り][通常論文]
     
    SACTE_5457 is secreted by Streptomyces sp. SirexAA-E, a highly cellulolytic actinobacterium isolated from a symbiotic community composed of insects, fungi, and bacteria. Here we report the 1.84 angstrom resolution crystal structure and functional characterization of SACTE_5457. This enzyme is a member of the glycosyl hydrolase family 46 and is composed of two -helical domains that are connected by an -helical linker. The catalytic residues (Glu74 and Asp92) are separated by 10.3 angstrom, matching the distance predicted for an inverting hydrolysis reaction. Normal mode analysis suggests that the connecting -helix is flexible and allows the domain motion needed to place active site residues into an appropriate configuration for catalysis. SACTE_5457 does not react with chitin, but hydrolyzes chitosan substrates with an approximate to 4-fold improvement in k(cat)/K-M as the percentage of acetylation and the molecular weights decrease. Analysis of the time dependence of product formation shows that oligosaccharides with degree of polymerization <4 are not hydrolyzed. By combining the results of substrate docking to the X-ray structure and end-product analysis, we deduce that SACTE_5457 preferentially binds substrates spanning the -2 to +2 sugar binding subsites, and that steric hindrance prevents binding of N-acetyl-d-glucosamine in the +2 subsite and may weakly interfere with binding of N-acetyl-d-glucosamine in the +1 subsites. A proposal for how these constraints account for the observed product distributions is provided. Proteins 2014; 82:1245-1257. (c) 2013 Wiley Periodicals, Inc.
  • Kai Deng, Taichi E. Takasuka, Richard Heins, Xiaoliang Cheng, Lai F. Bergeman, Jian Shi, Ryan Aschenbrener, Sam Deutsch, Seema Singh, Kenneth L. Sale, Blake A. Simmons, Paul D. Adams, Anup K. Singh, Brian G. Fox, Trent R. Northen
    ACS CHEMICAL BIOLOGY 9 7 1470 - 1479 2014年07月 [査読有り][通常論文]
     
    Glycoside hydrolases (GHs) are critical to cycling of plant biomass in the environment, digestion of complex polysaccharides by the human gut microbiome, and industrial activities such as deployment of cellulosic biofuels. High-throughput sequencing methods show tremendous sequence diversity among GHs, yet relatively few examples from the over 150,000 unique domain arrangements containing GHs have been functionally characterized. Here, we show how cell-free expression, bioconjugate chemistry, and surface-based mass spectrometry can be used to study glycoside hydrolase reactions with plant biomass. Detection of soluble products is achieved by coupling a unique chemical probe to the reducing end of oligosaccharides in a stable oxime linkage, while the use of C-13-labeled monosaccharide standards (xylose and glucose) allows quantitation of the derivatized glycans. We apply this oxime-based nanostructure-initiator mass spectrometry (NIMS) method to characterize the functional diversity of GHs secreted by Clostridium thermocellum, a model cellulolytic organism. New reaction specificities are identified, and differences in rates and yields of individual enzymes are demonstrated in reactions with biomass substrates. Numerical analyses of time series data suggests that synergistic combinations of mono- and multifunctional GHs can decrease the complexity of enzymes needed for the hydrolysis of plant biomass during the production of biofuels.
  • Katarzyna A. Gromek, Fabian P. Suchy, Hannah R. Meddaugh, Russell L. Wrobel, Loren M. LaPointe, Uyen B. Chu, John G. Primm, Arnold E. Ruoho, Alessandro Senes, Brian G. Fox
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 29 20333 - 20344 2014年07月 [査読有り][通常論文]
     
    Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[H-3]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.
  • Sungsoo Lim, Shishir P. S. Chundawat, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 98 1 - 9 2014年06月 [査読有り][通常論文]
     
    Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydratebinding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE_0237, the most abundantly expressed cellulase from SirexAA-E, was cloned into an Escherichia colt expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62 +/- 0.04 L/g) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose. (C) 2014 Elsevier Inc. All rights reserved.
  • Takasuka TE, Acheson JF, Bianchetti CM, Prom BM, Bergeman LF, Book AJ, Currie CR, Fox BG
    PloS one 9 4 e94166  2014年04月 [査読有り][通常論文]
     
    beta-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only beta-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl-and/ or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.
  • Makino S, Beebe ET, Markley JL, Fox BG
    Methods in molecular biology (Clifton, N.J.) 1091 161 - 178 2014年 [査読有り][通常論文]
  • Takasuka TE, Walker JA, Bergeman LF, Vander Meulen KA, Makino S, Elsen NL, Fox BG
    Methods in molecular biology (Clifton, N.J.) 1118 71 - 95 2014年 [査読有り][通常論文]
  • Beebe ET, Makino S, Markley JL, Fox BG
    Methods in molecular biology (Clifton, N.J.) 1140 117 - 135 2014年 [査読有り][通常論文]
  • Netra Joshi, Eric K. Hoobler, Steven Perry, Giovanni Diaz, Brian Fox, Theodore R. Holman
    BIOCHEMISTRY 52 45 8026 - 8035 2013年11月 [査読有り][通常論文]
     
    Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with their own hydroperoxide products. In this work, addition of both 13-(S)-hydroxy-(9Z,11E)-octadecadienoic acid [13-(S)-HODE] and 13-(S)-hydroperoxy-(6Z,9Z,11E)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the k(cat)/K-m substrate specificity ratio of arachidonic acid (AA) and gamma-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating k(cat)/K-M(AA) but inhibiting k(cat)/K-M(GLA), which indicates that the allosteric structural changes at the active site discriminate between the length and unsaturation differences of AA and GLA to achieve opposite kinetic effects. The substrate specificity ratio is further increased, 11-fold in total, with an increase in pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes.
  • Elizabeth J. Blaesi, Jessica D. Gardner, Brian G. Fox, Thomas C. Brunold
    BIOCHEMISTRY 52 35 6040 - 6051 2013年09月 [査読有り][通常論文]
     
    Cysteine dioxygenase (CDO) is a mononuclear nonheme iron(II)-dependent enzyme critical for maintaining appropriate cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys-Tyr crosslink near the active site, the mechanism by which it converts Cys and molecular oxygen to cysteine sulfinic acid is of broad interest. However, as of yet, direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O-2 to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual S = 1/2 {FeNO}(7) species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies as well as computational methods including density functional theory and semiempirical calculations. The NO adducts of Cys- and selenocysteine (Sec)-bound Fe(II)CDO exhibit virtually identical electronic properties; yet, CDO is unable to oxidize Sec. To explore the differences in reactivity between Cys- and Sec-bound CDO, the geometries and energies of viable O-2-bound intermediates were evaluated computationally, and it was found that a low-energy quintet-spin intermediate on the Cys reaction pathway adopts a different geometry for the Sec-bound adduct. The absence of a low-energy O-2 adduct for Sec. bound CDO is consistent with our experimental data and may explain why Sec is not oxidized by CDO.
  • Katarzyna A. Gromek, Hannah R. Meddaugh, Russell L. Wrobel, Fabian P. Suchy, Craig A. Bingman, John G. Primm, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 89 2 203 - 209 2013年06月 [査読有り][通常論文]
     
    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields similar to 3.5 mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling. (C) 2013 Elsevier Inc. All rights reserved.
  • Christopher M. Bianchetti, Connor H. Harmann, Taichi E. Takasuka, Gregory L. Hura, Kevin Dyer, Brian G. Fox
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 25 18574 - 18587 2013年06月 [査読有り][通常論文]
     
    Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core beta-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O-2-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response.
  • Yangrong Cao, David J. Aceti, Grzegorz Sabat, Junqi Song, Shin-ichi Makino, Brian G. Fox, Andrew F. Bent
    PLOS PATHOGENS 9 4 e1003313  2013年04月 [査読有り][通常論文]
     
    FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.
  • Khaled A. Aly, Emily T. Beebe, Chi H. Chan, Michael A. Goren, Carolina Sepulveda, Shin-ichi Makino, Brian G. Fox, Katrina T. Forest
    MICROBIOLOGYOPEN 2 1 94 - 104 2013年02月 [査読有り][通常論文]
     
    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-beta-D-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and Sadenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.
  • Pieper U, Schlessinger A, Kloppmann E, Chang GA, Chou JJ, Dumont ME, Fox BG, Fromme P, Hendrickson WA, Malkowski MG, Rees DC, Stokes DL, Stowell MH, Wiener MC, Rost B, Stroud RM, Stevens RC, Sali A
    Nature structural & molecular biology 20 2 135 - 138 2013年02月 [査読有り][通常論文]
     
    Given the recent successes in determining membrane-protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral a-helical transmembrane-domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human alpha-helical transmembrane domains from 26% to 58%, providing structure and function information not otherwise available.
  • Takasuka TE, Book AJ, Lewin GR, Currie CR, Fox BG
    Scientific reports 3 1030  2013年 [査読有り][通常論文]
  • Brian W. Jarecki, Shin-ichi Makino, Emily T. Beebe, Brian G. Fox, Baron Chanda
    SCIENTIFIC REPORTS 3 1040  2013年01月 [査読有り][通常論文]
     
    Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.
  • Aarthi Chandrasekaran, Kai Deng, Chung-Yan Koh, Taichi Takasuka, Lai F. Bergeman, Brian G. Fox, Paul D. Adams, Anup K. Singh
    CHEMICAL COMMUNICATIONS 49 48 5441 - 5443 2013年 [査読有り][通常論文]
     
    We describe a simple, multiplexed assay that integrates glycan synthesis, bioconjugation to microspheres, fluorescent chemical/biochemical detection and multiparameter flow cytometric analysis to screen activities of different families of carbohydrate-active enzymes.
  • Lucas J. Bailey, Justin F. Acheson, Jason G. McCoy, Nathaniel L. Elsen, George N. Phillips, Brian G. Fox
    BIOCHEMISTRY 51 6 1101 - 1113 2012年02月 [査読有り][通常論文]
     
    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric mu-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH2-benzoate and p-Br-benzoate showed a mu-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a pi-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 angstrom) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.
  • Byung Woo Han, Thomas E. Malone, Do Jin Kim, Craig A. Bingman, Hyun-Jung Kim, Brian G. Fox, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 79 11 3236 - 3241 2011年11月 [査読有り][通常論文]
  • Christopher M. Bianchetti, Nathaniel L. Elsen, Brian G. Fox, George N. Phillips
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 67 Pt 11 1345 - 1349 2011年11月 [査読有り][通常論文]
     
    Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into alpha-D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (alpha/alpha)(6)-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 angstrom resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.
  • Michael A. Goren, Brian G. Fox, James D. Bangs
    BIOCHEMISTRY 50 41 8853 - 8861 2011年10月 [査読有り][通常論文]
     
    The substrate selectivity of four Trypanosoma brucei sphingolipid syntheses was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSIS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC syntheses. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.
  • Hilary P. Beck, Michael DeGraffenreid, Brian Fox, John G. Allen, Yosup Rew, Stephen Schneider, Anne Y. Saiki, Dongyin Yu, Jonathan D. Oliner, Kevin Salyers, Qiuping Ye, Steven Olson
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 21 9 2752 - 2755 2011年05月 [査読有り][通常論文]
     
    Human murine double minute 2 (MDM2) is a negative regulator of p53, which plays an important role in cell cycle and apoptosis. We report several optimizations to the synthesis of the chromenotriazolopyrimidine series of MDM2-p53 protein-protein interaction inhibitors. Additionally, the in vitro and in vivo stability, pharmacokinetic properties and solubility were improved through N-substitution. (C) 2010 Elsevier Ltd. All rights reserved.
  • David A. Miller, Garret Suen, David Bruce, Alex Copeland, Jan-Feng Cheng, Chris Detter, Lynne A. Goodwin, Cliff S. Han, Loren J. Hauser, Miriam L. Land, Alla Lapidus, Susan Lucas, Linda Meincke, Sam Pitluck, Roxanne Tapia, Hazuki Teshima, Tanja Woyke, Brian G. Fox, Esther R. Angert, Cameron R. Currie
    JOURNAL OF BACTERIOLOGY 193 9 2357 - 2358 2011年05月 [査読有り][通常論文]
     
    Cellulosilyticum lentocellum DSM 5427 is an anaerobic, endospore-forming member of the Firmicutes. We describe the complete genome sequence of this cellulose-degrading bacterium, which was originally isolated from estuarine sediment of a river that received both domestic and paper mill waste. Comparative genomics of cellulolytic clostridia will provide insight into factors that influence degradation rates.
  • Emily T. Beebe, Shin-ichi Makino, Akira Nozawa, Yuko Matsubara, Ronnie O. Frederick, John G. Primm, Michael A. Goren, Brian G. Fox
    NEW BIOTECHNOLOGY 28 3 239 - 249 2011年04月 [査読有り][通常論文]
     
    The use of the Protemist X E, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2 mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KE,V). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins.
  • E. Sethe Burgie, Craig A. Bingman, Shin-ichi Makino, Gary E. Wesenberg, Xiaokang Pan, Brian G. Fox, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 79 4 1329 - 1336 2011年04月 [査読有り][通常論文]
  • Allison Riederer, Taichi E. Takasuka, Shin-ichi Makino, David M. Stevenson, Yury V. Bukhman, Nathaniel L. Elsen, Brian G. Fox
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 77 4 1243 - 1253 2011年02月 [査読有り][通常論文]
     
    A microarray study of chemostat growth on insoluble cellulose or soluble cellobiose has provided substantial new information on Clostridium thermocellum gene expression. This is the first comprehensive examination of gene expression in C. thermocellum under defined growth conditions. Expression was detected from 2,846 of 3,189 genes, and regression analysis revealed 348 genes whose changes in expression patterns were growth rate and/or substrate dependent. Successfully modeled genes included those for scaffoldin and cellulosomal enzymes, intracellular metabolic enzymes, transcriptional regulators, sigma factors, signal transducers, transporters, and hypothetical proteins. Unique genes encoding glycolytic pathway and ethanol fermentation enzymes expressed at high levels simultaneously with previously established maximal ethanol production were also identified. Ranking of normalized expression intensities revealed significant changes in transcriptional levels of these genes. The pattern of expression of transcriptional regulators, sigma factors, and signal transducers indicates that response to growth rate is the dominant global mechanism used for control of gene expression in C. thermocellum.
  • Euiyoung Bae, Eduard Bitto, Craig A. Bingman, Simon T. Allard, Gary E. Wesenberg, Russell L. Wrobel, Brian G. Fox, George N. Phillips
    JOURNAL OF THE KOREAN SOCIETY FOR APPLIED BIOLOGICAL CHEMISTRY 53 6 852 - 856 2010年12月 [査読有り][通常論文]
     
    Os09g0567400 codes for a hypothetical protein from Oryza sativa that is annotated as the "Histidine-containing phosphotransfer (Hpt) protein". Hpt domain is a protein module with a histidine residue mediating phosphotransfer reaction in the histidine-aspartate phosphorelay system. We report here the crystal structure and analysis of Os09g0567400.
  • Pil Seok Chae, Soren G. F. Rasmussen, Rohini R. Rana, Kamil Gotfryd, Richa Chandra, Michael A. Goren, Andrew C. Kruse, Shailika Nurva, Claus J. Loland, Yves Pierre, David Drew, Jean-Luc Popot, Daniel Picot, Brian G. Fox, Lan Guan, Ulrik Gether, Bernadette Byrne, Brian Kobilka, Samuel H. Gellman
    NATURE METHODS 7 12 1003 - U90 2010年12月 [査読有り][通常論文]
     
    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.
  • Jessica D. Gardner, Brad S. Pierce, Brian G. Fox, Thomas C. Brunold
    BIOCHEMISTRY 49 29 6033 - 6041 2010年07月 [査読有り][通常論文]
     
    Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(l I) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the cysteine analogue selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(H) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenascs.
  • Elitza S. Sevova, Michael A. Goren, Kevin J. Schwartz, Fong-Fu Hsu, John Turk, Brian G. Fox, James D. Bangs
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 27 20580 - 20587 2010年07月 [査読有り][通常論文]
     
    The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.
  • Gregory A. Zornetzer, Justinn Tanem, Brian G. Fox, John L. Markley
    BIOCHEMISTRY 49 3 470 - 477 2010年01月 [査読有り][通常論文]
     
    Acyl carrier proteins involved in fatty acid biosynthesis have been shown to exhibit a high degree of conformational flexibility, in that they are able to sequester fatty acid intermediates between 4 and 18 carbons in length. This flexibility has been observed in X-ray and NMR structures of acyl carrier proteins attached to different fatty acids. NMR studies comparing decanoyl-ACP and stearoyl-ACP indicated that ACP exhibits more dynamic motions when bound to longer fatty acids. We have used complementary chemical and NMR methods as an approach to Improving Our understanding of the effect of fatty acid length on the dynamics of acyl carrier protein. A chemical assay of the accessibility of the acyl thioester to solvent revealed a positive correlation between chain length and rate of hydrolysis. Surprisingly, this linear correlation was biphasic, with accelerated hydrolysis observed for fatty acids longer than 15 carbons. To further understand the motions associated with this acceleration, we collected (15)N relaxation dispersion data for 14:0-, 15:0-, and 16:0-ACP. The greatest dispersions were exhibited by residues that form the entrance to the fatty acid binding pocket. In addition, these dispersions were observed to increase with the length of the fatty acid. Because the exchange rates derived from fitting the data to a two-state model varied from residue to residue, a more complex motional model appears to be required to adequately explain the dynamics. Thus, acyl-ACP offers all interesting system for future investigations of complex protein motions oil the micro- and millisecond time scales.
  • Makino S, Goren MA, Fox BG, Markley JL
    Methods in molecular biology (Clifton, N.J.) 607 127 - 147 2010年 [査読有り][通常論文]
  • Yong Chang, David Mead, Vinay Dhodda, Phil Brumm, Brian G. Fox
    PROTEIN SCIENCE 18 11 2316 - 2325 2009年11月 [査読有り][通常論文]
     
    A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Delta(9) desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.
  • Mark E. Fuller, Kevin McClay, Jalal Hawari, Louise Paquet, Thomas E. Malone, Brian G. Fox, Robert J. Steffan
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 84 3 535 - 544 2009年09月 [査読有り][通常論文]
     
    The transformation of explosives, including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), by xenobiotic reductases XenA and XenB (and the bacterial strains harboring these enzymes) under both aerobic and anaerobic conditions was assessed. Under anaerobic conditions, Pseudomonas fluorescens I-C (XenB) degraded RDX faster than Pseudomonas putida II-B (XenA), and transformation occurred when the cells were supplied with sources of both carbon (succinate) and nitrogen (NH(4) (+)), but not when only carbon was supplied. Transformation was always faster under anaerobic conditions compared to aerobic conditions, with both enzymes exhibiting a O(2) concentration-dependent inhibition of RDX transformation. The primary degradation pathway for RDX was conversion to methylenedinitramine and then to formaldehyde, but a minor pathway that produced 4-nitro-2,4-diazabutanal (NDAB) also appeared to be active during transformation by whole cells of P. putida II-B and purified XenA. Both XenA and XenB also degraded the related nitramine explosives octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane. Purified XenB was found to have a broader substrate range than XenA, degrading more of the explosive compounds examined in this study. The results show that these two xenobiotic reductases (and their respective bacterial strains) have the capacity to transform RDX as well as a wide variety of explosive compounds, especially under low oxygen concentrations.
  • Lucas J. Bailey, Brian G. Fox
    BIOCHEMISTRY 48 38 8932 - 8939 2009年09月 [査読有り][通常論文]
     
    A diiron hydroxylase reaction typically begins by combination of O(2) with a diferrous center to form reactive intermediates capable of hydrocarbon hydroxylation. In this natural cycle, reducing equivalents are provided by specific interactions with electron transfer proteins. The biological process can be bypassed by combining H(2)O(2) with a diferric center, i.e., peroxide-shunt catalysis. Here we show that toluene 4-monooxygenase has a peroxide-shunt reaction that is similar to 600-fold slower than catalysis driven by biological electron transfer. However, the toluene 4-monooxygenase hydroxylase-effector protein complex was stable in the presence of 300 mM H(2)O(2), suggesting overall benign effects of the exogenous oxidant on active site structure and function. The X-ray structure of the toluene 4-monooxygenase hydroxylase-effect or protein complex determined from crystals soaked in H(2)O(2) revealed a bound diatomic molecule, assigned to a cis-mu-1,2-peroxo bridge. This peroxo species resides in an active site position adjacent to the hydrogen-bonding substructure established by effector protein binding and faces into the distal cavity where substrate must bind during regiospecific aromatic ring hydroxylation catalysis. These results provide a new structural benchmark for how activated intermediates may be formed and dispatched during diiron hydroxylase catalysis.
  • Eduard Bitto, Craig A. Bingman, Lenka Bittova, Ronnie O. Frederick, Brian G. Fox, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 76 2 477 - 483 2009年08月 [査読有り][通常論文]
     
    Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF-hand proteins represent a superfamily of calcium-binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa-EF-hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X-ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium-free form at 2.1-angstrom resolution. Secretagogin consist, of the three globular domains each of which contains a pair of EF-hand motifs. The domains are arranged into a V-shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium-loaded calbindin D(28K) revealed a striking difference in the spatial arrangement of their domains, which involves similar to 180 degrees rotation of the first globular domain with respect to the module formed by the remaining domains.
  • Nathaniel L. Elsen, Lucas J. Bailey, Andrew D. Hauser, Brian G. Fox
    BIOCHEMISTRY 48 18 3838 - 3846 2009年05月 [査読有り][通常論文]
     
    The active site residue Thr-201 in toluene 4-monooxygenase hydroxylase (T4moH) has a structural counterpart in the active sites of all diiron monooxygenases. Thus, our previous finding that mutation of this residue to Ala, Gly, or Ser had no impact on steady-state catalysis or coupling was surprising. In this work., we provide kinetic, biochemical, and structural evidence that one role of Thr-201 may be to stabilize a peroxo-level intermediate during enzyme catalysis. During reactions in the absence of substrate, T201 T4moH slowly consumed O(2) but only a negligible amount of H(2)O(2) was released. In contrast, T201A T4moH gave stoichometric release of H(2)O(2) during reaction in the absence of substrate. Both enzyme isoforms were tightly coupled during steady-state catalysis with saturating toluene and other optimal substrates and exhibited near-identical kinetic parameters. However, rapid mix single-turnover studies showed that T201A T4moH had a faster first-order rate constant for product formation than T201 T4moH did. Comparison of X-ray crystal structures of resting and reduced T201A T4moH in complex with T4moD With comparable structures of T201 T4moHD revealed changes in the positions of several key active site residues relative to the comparable structures of T201 T4moH with T4moD. This combination of catalytic and structural studies offers important new insight into the role of the role of conserved Thr-201, and its contributions to the catalytic reaction cycle.
  • Markley JL, Aceti DJ, Bingman CA, Fox BG, Frederick RO, Makino S, Nichols KW, Phillips GN Jr, Primm JG, Sahu SC, Vojtik FC, Volkman BF, Wrobel RL, Zolnai Z
    Journal of structural and functional genomics 10 2 165 - 179 2009年04月 [査読有り][通常論文]
  • Fox BG, Blommel PG
    Current protocols in protein science Chapter 5 Unit 5.23  2009年04月 [査読有り][通常論文]
  • Jason G. McCoy, Lucas J. Bailey, Yi Han Ng, Craig A. Bingman, Russell Wrobel, Andreas P. M. Weber, Brian G. Fox, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 74 2 368 - 377 2009年02月 [査読有り][通常論文]
     
    An enzyme with sarcosine dimethylglycine methyltransferase (SDMT) activity has been identified in the thermophilic eukaryote, Galdieria sulphuraria The crystal structure of the enzyme, solved to a resolution of 1.95 angstrom, revealed a fold highly similar to that of mycolic acid synthases. The k(cat), and apparent Km values were 64.3 min(-1) and 2.0 mM for sarcosine and 85.6 min(-1) and 2.8 mM for dimethylglycine, respectively. Apparent Km values of S-adenosylmethionine were 144 and 150 mu M for sarcosine and dimethylglycine, respectively, and the enzyme melting temperature was 61.1 degrees C. Modeling of cofactor binding in the active site based on the structure of methoxy mycolic acid synthase 2 revealed a number of conserved interactions within the active site.
  • Eduard Bitto, Craig A. Bingman, Lenka Bittova, Norma L. Houston, Rebecca S. Boston, Brian G. Fox, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 74 1 61 - 71 2009年01月 [査読有り][通常論文]
     
    The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 angstrom resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alpha beta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.
  • Blommel PG, Martin PA, Seder KD, Wrobel RL, Fox BG
    Methods in molecular biology (Clifton, N.J.) 498 55 - 73 2009年 [査読有り][通常論文]
  • Michael A. Goren, Akira Nozawa, Shin-ichi Makino, Russell L. Wrobel, Brian G. Fox
    GUIDE TO PROTEIN PURIFICATION, SECOND EDITION 463 647 - 673 2009年 [査読有り][通常論文]
     
    Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.
  • Michael A. Goren, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 62 2 171 - 178 2008年12月 [査読有り][通常論文]
     
    A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed. (c) 2008 Elsevier Inc. All rights reserved.
  • Lucas J. Bailey, Jason G. Mccoy, George N. Phillips, Brian G. Fox
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 49 19194 - 19198 2008年12月 [査読有り][通常論文]
     
    Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 angstrom from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.
  • David J. Aceti, Eduard Bitto, Alexander F. Yakunin, Michael Proudfoot, Craig A. Bingman, Ronnie O. Frederick, Hassan K. Sreenath, Frank C. Vojtik, Russell L. Wrobel, Brian G. Fox, John L. Markley, George N. Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 73 1 241 - 253 2008年10月 [査読有り][通常論文]
     
    The crystal structure of the protein product of the gene locus At1g05000, a hypothetical protein from A. thaliana, was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 20.4% (R-free = 24.9%) at 3.3 angstrom. The protein adopts the alpha/beta fold found in cysteine phosphatases, a superfamily of phosphatases that possess a catalytic cysteine and form a covalent thiol-phosphate intermediate during the catalytic cycle. In At1g05000, the analogous cysteine (Cys(150)) is located at the bottom of a positively-charged pocket formed by residues that include the conserved arginine (Arg(156)) of the signature active site motif, HCxxGxxRT. Of 74 model phosphatase substrates tested, purified recombinant At1g05000 showed highest activity toward polyphosphate (poly-P12-13) and deoxyribo- and ribonucleoside triphosphates, and less activity toward phosphoenolpyruvate, phosphotyrosine, phosphotyrosine-containing peptides, and phosphatidyl inositols. Divalent metal cations were not required for activity and had little effect on the reaction.
  • Eduard Bitto, Craig A. Bingman, Lenka Bittova, Dmitry A. Kondrashov, Ryan M. Bannen, Brian G. Fox, John L. Markley, George N. Phillips
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 44 30184 - 30192 2008年10月 [査読有り][通常論文]
     
    Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0 angstrom crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX(9-13)FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals.
  • Yong Chang, Gary E. Wesenberg, Craig A. Bingman, Brian G. Fox
    JOURNAL OF BACTERIOLOGY 190 20 6686 - 6696 2008年10月 [査読有り][通常論文]
     
    DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Delta(9) desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His(6) or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that similar to 10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.
  • Elena J. Levin, Nathaniel L. Elsen, Kory D. Seder, Jason G. McCoy, Brian G. Fox, George N. Phillips
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 64 Pt 9 933 - 940 2008年09月 [査読有り][通常論文]
     
    The 2.07 angstrom resolution X-ray crystal structure of a soluble Rieske-type ferredoxin from Mus musculus encoded by the gene Mm. 266515 is reported. Although they are present as covalent domains in eukaryotic membrane oxidase complexes, soluble Rieske-type ferredoxins have not previously been observed in eukaryotes. The overall structure of the mouse Rieske-type ferredoxin is typical of this class of iron-sulfur proteins and consists of a larger partial beta-barrel domain and a smaller domain containing Cys57, His59, Cys80 and His83 that binds the [2Fe-2S] cluster. The S atoms of the cluster are hydrogen-bonded by six backbone amide N atoms in a pattern typical of membrane-bound high-potential eukaryotic respiratory Rieske ferredoxins. However, phylogenetic analysis suggested that the mouse Rieske-type ferredoxin was more closely related to bacterial Rieske-type ferredoxins. Correspondingly, the structure revealed an extended loop most similar to that seen in Rieske-type ferredoxin subunits of bacterial aromatic dioxygenases, including the positioning of an aromatic side chain (Tyr85) between this loop and the [2Fe-2S] cluster. The mouse Rieske-type ferredoxin was shown to be capable of accepting electrons from both eukaryotic and prokaryotic oxidoreductases, although it was unable to serve as an electron donor for a bacterial monooxygenase complex. The human homolog of mouse Rieske-type ferredoxin was also cloned and purified. It behaved identically to mouse Rieske-type ferredoxin in all biochemical characterizations but did not crystallize. Based on its high sequence identity, the structure of the human homolog is likely to be modeled well by the mouse Rieske-type ferredoxin structure.
  • Jennifer K. Schwartz, Pin-pin Wei, Kevin H. Mitchell, Brian G. Fox, Edward I. Solomon
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 130 22 7098 - 7109 2008年06月 [査読有り][通常論文]
     
    Multicomponent monooxygenases, which carry out a variety of highly specific hydroxylation reactions, are of great interest as potential biocatalysts in a number of applications. These proteins share many similarities in structure and show a marked increase in O-2 reactivity upon addition of an effector component. In this study, circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD have been used to gain spectroscopic insight into the Fe(II)Fe(II) active site in the hydroxylase component of Toluene-4 monoxygenase (T4moH) and the complex of T4moH bound by its effector protein, T4moD. These results have been correlated to spectroscopic data and density functional theory (DFT) calculations on MmoH and its interaction with MmoB. Together, these data provide further insight into the geometric and electronic structure of these biferrous active sites and, in particular, the perturbation associated with component B/D binding. It is found that binding of the effector protein changes the geometry of one iron center and orientation of its redox active orbital to accommodate the binding of O-2 in a bridged structure for efficient 2-electron transfer that can form a peroxo intermediate.
  • Pablo Sobrado, Michael A. Goren, Declan James, Carissa K. Amundson, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 58 2 229 - 241 2008年04月 [査読有り][通常論文]
     
    A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of similar to 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed. (C) 2007 Elsevier Inc. All rights reserved.
  • Brian G. Fox, Celia Goulding, Michael G. Malkowski, Lance Stewart, Ashley Deacon
    NATURE METHODS 5 2 129 - 132 2008年02月 [査読有り][通常論文]
     
    The Protein Structure Initiative (PSI), funded by the US National Institutes of Health (NIH), provides a framework for the development and systematic evaluation of methods to solve protein structures. Although the PSI and other structural genomics efforts around the world have led to the solution of many new protein structures as well as the development of new methods, methodological bottlenecks still exist and are being addressed in this ' production phase' of PSI.
  • Lucas J. Bailey, Nathaniel L. Elsen, Brad S. Pierce, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 57 1 9 - 16 2008年01月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21 (DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded similar to 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained I mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed similar to 1 mol of the S = 1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent KM- and apparent k(cat)-values of 0.15 mu M and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations. (c) 2007 Elsevier Inc. All rights reserved.
  • Frederick RO, Bergeman L, Blommel PG, Bailey LJ, McCoy JG, Song J, Meske L, Bingman CA, Riters M, Dillon NA, Kunert J, Yoon JW, Lim A, Cassidy M, Bunge J, Aceti DJ, Primm JG, Markley JL, Phillips GN Jr, Fox BG
    Journal of structural and functional genomics 8 4 153 - 166 2007年12月 [査読有り][通常論文]
  • Pan X, Wesenberg GE, Markley JL, Fox BG, Phillips GN Jr, Bingman CA
    Journal of structural and functional genomics 8 4 209 - 216 2007年12月 [査読有り][通常論文]
  • Paul G. Blommel, Brian G. Fox
    PROTEIN EXPRESSION AND PURIFICATION 55 1 53 - 68 2007年09月 [査読有り][通常論文]
     
    Tobacco etch virus NIa proteinase (TEV protease) is an important too] for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of similar to 400 mg/L of expression culture (similar to 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (similar to 12 mg pure protease fusion per gram of E. coli cell paste) are also reported. (C) 2007 Elsevier Inc. All rights reserved.
  • Phillips GN Jr, Fox BG, Markley JL, Volkman BF, Bae E, Bitto E, Bingman CA, Frederick RO, McCoy JG, Lytle BL, Pierce BS, Song J, Twigger SN
    Journal of structural and functional genomics 8 2-3 73 - 84 2007年09月 [査読有り][通常論文]
  • Brad S. Pierce, Jessica D. Gardner, Lucas J. Bailey, Thomas C. Brunold, Brian G. Fox
    BIOCHEMISTRY 46 29 8569 - 8578 2007年07月 [査読有り][通常論文]
     
    Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the N epsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O-2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of L-cysteine prior to NO (and presumably O-2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of L-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}(7) (S)=3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of similar to 4, similar to 4, and similar to 2, respectively. However, upon addition of NO to CDO in the presence of substrate L-cysteine, a low-spin {FeNO}(7) (S=1/2) signal that accounts for similar to 85% of the iron within the enzyme develops. Similar {FeNO}(7) (S=1/ 2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of L-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}(7) signals (S=1/2 or 3/ 2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO} 7 (S=1/ 2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}(7)) is a result of the bidentate thiol/ amine coordination of L-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO} 7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}(7) (S=1/2) iron-nitrosyl complexes.
  • Paul G. Blommel, Katie J. Becker, Petar Duvnjak, Brian G. Fox
    BIOTECHNOLOGY PROGRESS 23 3 585 - 598 2007年05月 [査読有り][通常論文]
     
    The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 mu g/mL of expression culture were identified.
  • Nathaniel L. Elsen, Luke A. Moe, Lea A. McMartin, Brian G. Fox
    BIOCHEMISTRY 46 4 976 - 986 2007年01月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase catalyzes the NADH- and O-2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkeholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent K-M only, while D82A/D83A T4moC gave alterations in both apparent k(cat) and K-M. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.
  • Ashok Marwah, F. Enrique Gomez, Padma Marwah, James M. Ntambi, Brian G. Fox, Henry Lardy
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 456 1 1 - 7 2006年12月 [査読有り][通常論文]
     
    Dehydroepiandrosterone is known to depress fatty acid formation in differentiating 3T3-L1 adipocytes. The metabolism of dehydroepiandrosterone and four of its natural metabolites in differentiating adipocytes was studied by liquid chromatography-mass spectrometry. Adipocytes rapidly converted dehydroepiandrosterone to androst-5-ene-3p,17 beta-diol. 7 alpha-Hydroxy-DHEA was interconverted with 7-oxo-DHEA and 7 beta-hydroxy-DHEA and the corresponding 17 beta reduced products. Dehydroepiandrosterone and its derivatives were detected only in the culture medium suggesting that dehydroepiandrosterone is metabolized via enzymes located in close proximity to, or that are integral parts of the cell membrane. Alternatively, there may be efficient mechanisms at play for extrusion of the steroids to the aqueous media rather than being retained in the lipid-rich cell. An interesting aspect of the study was finding androstenediol as the major metabolite of dehydroepiandrosterone. Androst-5-ene-3 beta,17 beta-diol has been implicated in prostate cancer. The contribution of adipose cells to the circulating supply of androst-5-ene-3p,17 beta-diol may therefore be considered in managing prostate cancer. (c) 2006 Elsevier Inc. All rights reserved.
  • Yong Chang, Brian G. Fox
    BIOCHEMISTRY 45 45 13476 - 13486 2006年11月 [査読有り][通常論文]
     
    DesA3 is a membrane-bound stearoyl- CoA Delta(9)-desaturase that produces oleic acid, a precursor of mycobacterial membrane phospholipids and triglycerides. The sequence of DesA3 is homologous with those of other membrane desaturases, including the presence of the eight-His motif proposed to bind the diiron center active site. This family of desaturases function as multicomponent complexes and thus require electron transfer proteins for efficient catalytic turnover. Here we present evidence that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. For these studies, Rv3230c was expressed as a partially soluble protein in Escherichia coli; recombinant DesA3 was expressed in Mycobacterium smegmatis as a catalytically active membrane protein. The addition of E. coli lysates containing Rv3230c to lysates of M. smegmatis expressing DesA3 gave strong conversion of [1-C-14]-18:0-CoA to [1-C-14]-cis-Delta(9)-18:1-CoA and of [1-C-14]-16:0-CoA to [1-C-14]-cis-Delta(9)-16:1-CoA. Both M. tuberculosis proteins were required for reconstitution of activity, as various combinations of control lysates lacking either Rv3230c or DesA3 gave minimal or no activity. Furthermore, the specificity of interaction between Rv3230c and DesA3 was implied by the inability of other related redox systems to substitute for Rv3230c. The reconstituted activity was dependent upon the presence of NADPH, could be saturated by increasing the amount of Rv3230c added, and was also sensitive to the salt concentration in the buffer. The results are consistent with the formation of a protein-protein complex, possibly with electrostatic character. This work defines a multiprotein, acyl-CoA desaturase complex from M. tuberculosis H37Rv to minimally consist of a soluble Rv3230c reductase and integral membrane DesA3 desaturase. Further implications of this finding relative to the properties of other multiprotein ironenzyme complexes are discussed.
  • PG Blommel, PA Martin, RL Wrobel, E Steffen, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 47 2 562 - 570 2006年06月 [査読有り][通常論文]
     
    The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated. (c) 2005 Elsevier Inc. All rights reserved.
  • LA Moe, CA Bingman, GE Wesenberg, GN Phillips, BG Fox
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 62 Pt 5 476 - 482 2006年05月 [査読有り][通常論文]
     
    The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe-2S](2+) state at a resolution of 1.48 angstrom using single-wavelength anomalous dispersion phasing with the [2Fe-2S] center. The structure consists of ten beta-strands arranged into the three antiparallel beta-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe--2S] centre, which displaces a loop containing the conserved Pro81 by similar to 8 angstrom away from the [2Fe-2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe-2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis-dihydrodiol-forming dioxygenases.
  • LA Moe, LA McMartin, BG Fox
    BIOCHEMISTRY 45 17 5478 - 5485 2006年05月 [査読有り][通常論文]
     
    A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region to contain a tetraCys motif and treatment with biarsenical fluorescein. Fluorescence anisotropy was used to study the protein-protein interactions among various combinations of the four components of the complex. Binding interactions were detected between T4moD and the hydroxylase component T4moH [K-D value of 83 nM for interaction with the alpha beta gamma protomer] and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC (KD value of 78 nM). No binding interactions were detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF was able to disrupt binding between T4moC and T4moD. The detected binding interactions suggest an intermediary electron transfer complex between T4moC and T4moD that excludes T4moF. The results indicate that specialization of effector protein function may include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.
  • GA Zornetzer, RD White, JL Markley, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 46 2 446 - 455 2006年04月 [査読有り][通常論文]
     
    Acyl carrier proteins (ACPs) are important protein cofactors in fatty acid biosynthesis, but their acylated forms have not been well-studied. To permit detailed nuclear magnetic resonance studies of acylated spinach ACP isoform I, we have developed a new expression plasmid for recombinant production of the apo-protein and modified protocols for purifying the protein product and acylating it to form acyl-ACP. To solve plasmid stability problems associated with growth in minimal media, the ampicillin resistance gene from pSACP-2a was replaced with the tetA(C) gene from pBR322. The resulting plasmid, pSACP-2t, supported overexpression of apo-ACP in Escherichia coli BL21 (DE3) cells in M9 medium containing (NH4Cl)-N-15 as the sole nitrogen source. Apo-ACP was purified to homogeneity by means of polyethylene glycol precipitation and anion exchange. Two in vitro synthetic routes were used to produce acyl-ACPs. In one route, apo-ACP was converted to the holo form and the acyl form by a published protocol that employs a discrete enzymatic reaction for each step. As an alternative route to produce decanoyl-ACP, apo-ACP was directly converted to the acyl form by using holo-ACP synthase along with the non-natural substrate decanoyl-CoA. Two-dimensional H-1-N-15 NMR spectroscopy of decanoyl-ACP and stearoyl-ACP revealed that changes in the length of the covalently attached fatty acid do not affect the secondary structure of the protein but do influence the local conformation and dynamics. (c) 2005 Elsevier Inc. All rights reserved.
  • P Sobrado, KS Lyle, SP Kaul, MM Turco, Arabshahi, I, A Marwah, BG Fox
    BIOCHEMISTRY 45 15 4848 - 4858 2006年04月 [査読有り][通常論文]
     
    Stearoyl-acyl carrier protein desaturase (Delta 9D) catalyzes the 02 and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta 9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta 9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta 9D decreased the k(cat)/K-M for Fd by 4-, 22-, and 2400-K60A respectively, as compared to wt Delta 9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K-M for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K-M for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta 9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta 9D and other protein-protein complexes.
  • GA Zornetzer, BG Fox, JL Markley
    BIOCHEMISTRY 45 16 5217 - 5227 2006年04月 [査読有り][通常論文]
     
    Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus, it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4'-phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo- and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0- and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4'-phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0- and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach ACP and suggest how the protein changes to accommodate its myriad binding partners.
  • JG McCoy, LJ Bailey, E Bitto, CA Bingman, DJ Aceti, BG Fox, GN Phillips
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 9 3084 - 3089 2006年02月 [査読有り][通常論文]
     
    Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 angstrom. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical beta-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.
  • LA Moe, BG Fox
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 338 1 240 - 249 2005年12月 [査読有り][通常論文]
     
    This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of (18)O(2) and (18)OH(2) to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with k(cat) and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O(2), while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed. (c) 2005 Elsevier Inc. All rights reserved.
  • BG Fox, TE Malone, KA Johnson, SE Madson, M Aceti, CA Bingman, PG Blommel, B Buchan, B Burns, J Cao, C Cornilescu, J Doreleijers, J Ellefson, R Frederick, H Geetha, D Hruby, WB Jeon, T Kimball, J Kunert, JL Markley, C Newman, A Olson, FC Peterson, GN Phillips, J Primm, B Ramirez, NS Rosenberg, M Runnels, K Seder, J Shaw, DW Smith, H Sreenath, J Song, MR Sussman, S Thao, D Troestler, E Tyler, R Tyler, E Ulrich, D Vinarov, F Vojtik, BF Volkman, G Wesenberg, RL Wrobel, J Zhang, Q Zhao, Z Zolnai
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 61 1 206 - 208 2005年10月 [査読有り][通常論文]
  • E Bertrand, R Sakai, E Rozhkova-Novosad, L Moe, BG Fox, JT Groves, RN Austin
    JOURNAL OF INORGANIC BIOCHEMISTRY 99 10 1998 - 2006 2005年10月 [査読有り][通常論文]
     
    Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPol expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximate to 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximate to 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximate to 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AIkB. (c) 2005 Elsevier Inc. All rights reserved.
  • DH Dyer, KS Lyle, Rayment, I, BG Fox
    PROTEIN SCIENCE 14 6 1508 - 1517 2005年06月 [査読有り][通常論文]
     
    Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(similar to 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA I and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 angstrom resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta 9 desaturase from castor plant with an rms difference 1.42 angstrom. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.
  • RC Tyler, DJ Aceti, CA Bingman, CC Cornilescu, BG Fox, RO Frederick, WB Jeon, MS Lee, CS Newman, FC Peterson, GN Phillips, MN Shahan, S Singh, JK Song, HK Sreenath, EM Tyler, EL Ulrich, DA Vinarov, FC Vojtik, BF Volkman, RL Wrobel, Q Zhao, JL Markley
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 59 3 633 - 643 2005年05月 [査読有り][通常論文]
     
    We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of N-15-labeled proteins to be analyzed by H-1-N-15 NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-firee technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NAIR sample too dilute. Those targets that reached the goal of analysis by H-1-N-15 NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+ (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-firee platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination. (c) 2005 Wiley-Liss, Inc.
  • DJ Baumler, KC Jeong, BG Fox, JF Banfield, CW Kaspar
    RESEARCH IN MICROBIOLOGY 156 4 492 - 498 2005年05月 [査読有り][通常論文]
     
    Growth of the acidophilic archaeon, '' Ferroplasma acidarmanus '' strain fer1, in a laboratory medium (primary constituents, FeSO4 72 mM and 0.02% yeast extract) is minimal. A survey of the annotated genome revealed metabolic transporters for Ni2+, sugars, and amino acids. Accordingly, the concentration of yeast extract was increased to 0.1% and the addition of 2 mM Ni(NH4)(2)(SO4)(2) significantly enhanced the cultivation of strain fer1. The maximum optical density in the modified fer1 medium (mfer) was OD492 = 0.27 with 10(10) viable cells/ml as determined by a most-probable-number method, which exceeds previously reported viable cells/ml by > 100-fold. Strain fer1 displayed chemolithotrophic growth with Fe2+ in mfer containing 100 mM FeSO4 or FeCl2. In the absence of Fe2+, heterotrophic growth occurred with one of the following salts (100 mM): ZnSO4, MnSO4, MgSO4, (NH4)(2)SO4, or Fe-2(SO4)(3), and did not occur with (100 mM): ZnCl2, MnCl2, MgCl2, NH4Cl, or FeCl3. Escaping headspace gas from strain fer1 cultures formed a precipitate in a zinc acetate trap. Sulfide was absent in the precipitate but zinc and sulfur were detected. These data demonstrate that SO4 is required for heterotrophic growth of strain fer1 and may have a role in the global sulfur cycle. (c) 2005 Elsevier SAS. All rights reserved.
  • GT Lountos, KH Mitchell, JM Studts, BG Fox, AM Orville
    BIOCHEMISTRY 44 19 7131 - 7142 2005年05月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (&UDelta; N4-) or 10 (&UDelta; N10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-&ANGS; resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 &ANGS; for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from &SIM; 1.8 &ANGS; to greater than 6 &ANGS;. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) &ANGS; based on the R-value (R free) for the highest resolution &UDelta; N4-T4moD structure to 0.211 (0.196) &ANGS; for the native T4moD structure. Catalytic studies of the &UDelta; N4-, &UDelta; N7-, and &UDelta; N10- variants of T4moD show statistically insignificant changes in k(cat), K-m, k(cat)/K-m, and K-1 relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the &SIM; 33 residue N-terminal region for catalytic activity.
  • E Bitto, CA Bingman, STM Allard, GE Wesenberg, DJ Aceti, RL Wrobel, RO Frederick, H Sreenath, FC Vojtik, WB Jeon, CS Newman, J Primm, MR Sussman, BG Fox, JL Markley, GN Phillips
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 Pt 5 469 - 472 2005年05月 [査読有り][通常論文]
  • HK Sreenath, CA Bingman, BW Buchan, KD Seder, BT Burns, HV Geetha, WB Jeon, FC Vojtik, DJ Aceti, RO Frederick, GN Phillips, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 40 2 256 - 267 2005年04月 [査読有り][通常論文]
     
    Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The larp-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L-1 selenomethionine, salts and trace metals, other amino acids including 10 mg L-1 of methionine, vitamins except vitamin B-12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take similar to 24 h. Culture growth in the autoinduction medium gave an average final optical density at 600 nm of similar to 6 and an average wet cell mass yield of similar to 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was similar to 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a similar to 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing. (c) 2005 Elsevier Inc. All rights reserved.
  • RC Tyler, HK Sreenath, S Singh, DJ Aceti, CA Binginan, JL Markley, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 40 2 268 - 278 2005年04月 [査読有り][通常論文]
     
    Protocols have been developed and applied for the high-throughput production of [U-N-15]- or [U-C-13-, U-15 N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B-12,, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of similar to 5, an average wet cell mass yield of -9.5 g L-1, and an average yield of similar to 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both N-15 and C-13 at levels greater than 95%. H-1-N-15 heteronuclear single quantum correlation spectroscopy as well as, C-13; N-15-edited spectroscopy showed that the purified [U-(15) N]- and [U-C-13, U-(15) N]-labeled proteins were suitable for structure analysis. (c) 2005 Elsevier Inc. All rights reserved.
  • Stolc, V, MP Samanta, W Tongprasit, H Sethi, SD Liang, DC Nelson, A Hegeman, C Nelson, D Rancour, S Bednarek, EL Ulrich, Q Zhao, RL Wrobel, CS Newman, BG Fox, GN Phillips, JL Markley, MR Sussman
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 12 4453 - 4458 2005年03月 [査読有り][通常論文]
     
    Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.
  • NM Nesbitt, C Baleanu-Gogonea, RM Cicchillo, K Goodson, DF Iwig, JA Broadwater, JA Haas, BG Fox, SJ Booker
    PROTEIN EXPRESSION AND PURIFICATION 39 2 269 - 282 2005年02月 [査読有り][通常論文]
     
    Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several a-keto acids. In its functional form. it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit." resulting in a long ",swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms. into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg(-1) indicating a turnover number of similar to0.2 s(-1) and the apparent K-m values for ocianoyl-ACP and apo-H protein are 10.2 +/- 4.4 and 13.2 +/- 2.9 muM, respectively. (C) 2004 Elsevier Inc. All rights reserved.
  • TE Malone, SE Madson, RL Wrobel, WB Jeon, NS Rosenberg, KA Johnson, CA Bingman, DW Smith, GN Phillips, JL Markley, BG Fox
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 58 1 243 - 245 2005年01月 [査読有り][通常論文]
  • PG Blommel, BG Fox
    ANALYTICAL BIOCHEMISTRY 336 1 75 - 86 2005年01月 [査読有り][通常論文]
     
    A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. The recombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and different protease recognition sites were created and tested for reaction with commercial proteases commonly used to process recombinant fusion proteins. In each case, proteolysis of a single susceptible peptide bond could be monitored in real time and with sufficient data quality to allow numerical analysis of proteolysis reaction kinetics. Measurement of proteolysis extent using fluorescence anisotropy is shown to be comparable to densitometry measurements made on denaturing polyacrylamide gels but with the added advantages implicit in a time-resolved measurement, quantification by a spectroscopic measurement, and facile extensibility to high-throughput formats. The assay was also demonstrated as a general tool for monitoring proteolysis of multidomain fusion proteins containing an internal protease site such as are being created in structural genomics studies worldwide. (C) 2004 Elsevier Inc. All rights reserved.
  • Jeon WB, Aceti DJ, Bingman CA, Vojtik FC, Olson AC, Ellefson JM, McCombs JE, Sreenath HK, Blommel PG, Seder KD, Burns BT, Geetha HV, Harms AC, Sabat G, Sussman MR, Fox BG, Phillips GN Jr
    Journal of structural and functional genomics 6 2-3 143 - 147 2005年 [査読有り][通常論文]
  • DW Smith, KA Johnson, CA Bingman, DJ Aceti, PG Blommel, RL Wrobel, RO Frederick, Q Zhao, H Sreenath, BG Fox, BF Volkman, WB Jeon, CS Newman, EL Ulrich, AD Hegeman, T Kimball, S Thao, MR Sussman, JL Markley, GN Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 57 4 854 - 857 2004年12月 [査読有り][通常論文]
  • L Skjeldal, FC Peterson, JF Doreleijers, LA Moe, JD Pikus, WM Westler, JL Markley, BF Volkman, BG Fox
    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 9 8 945 - 953 2004年12月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O-2-dependent hydroxylation of toluene to form p-cresol. The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D H-1, C-13, and N-15 NMR data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle (phi, psi) restraints. The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality. With exclusion of ill-defined N- and C-terminal segments (Ser2; His111-Ser112) and residues near to the [2Fe-2S] cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 Angstrom for the backbone and 1.60 Angstrom for all non-hydrogen atoms. The T4moC structure consists of 10 beta-strands arranged in the three anti-parallel beta-sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S-gamma of Cys45 and Cys64 and the N-delta1 of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. H-1 - N-15 HSQC measurements show that both His47-N-epsilon2 and His67-N-epsilon2 are protonated at the pH of the NMR experiments. Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family.
  • LA Moe, ZB Hu, DY Deng, RN Austin, JT Groves, BG Fox
    BIOCHEMISTRY 43 50 15688 - 15701 2004年12月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO (T4moH) with different regiospecificities for toluene hydroxylation (k(cat) approximate to 1.9-2.3 s(-1) and coupling efficiency approximate to 81-96%) revealed similar catalytic parameters for norcarane oxidation (k(cat) approximate to 0.3-0.5 s(-1) and coupling efficiency approximate to 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with similar to50-fold slower k(cat) values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O-2 in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H2O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.
  • CA Bingman, KA Johnson, FC Peterson, RO Frederick, Q Zhao, S Thao, BG Fox, BF Volkman, WB Jeon, DW Smith, CS Newman, EL Ulrich, A Hegeman, MR Sussman, JL Markley, GN Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 57 1 218 - 220 2004年10月 [査読有り][通常論文]
  • PG Blommel, DW Smith, CA Bingman, DH Dyer, Rayment, I, HM Holden, BG Fox, GN Phillips
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 57 1 221 - 222 2004年10月 [査読有り][通常論文]
  • AM Orville, L Manning, DS Blehert, BG Fox, GH Chambliss
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 60 Pt 7 1289 - 1291 2004年07月 [査読有り][通常論文]
     
    Single crystals have been obtained of xenobiotic reductase B ( XenB), a flavoenzyme isolated and cloned from Pseudomonas fluorescens I-C. The enzyme catalyzes the NADPH-dependent elimination of nitrite from nitroglycerin with an approximately fivefold kinetic preference for the middle nitro group, primarily yielding 1,3-dinitroglycerol. X-ray diffraction data sets have been collected from native crystals to 2.3 Angstrom resolution. The space group is P4(1)2(1)2, with unit-cell parameters a = b = 140, c = 95.6 Angstrom. The asymmetric unit is likely to contain at least two XenB molecules ( V-M = 3.1 Angstrom(3) Da(-1), 60% solvent) and a molecular-replacement solution has been determined in order to solve the structure.
  • BG Fox, KS Lyle, CE Rogge
    ACCOUNTS OF CHEMICAL RESEARCH 37 7 421 - 429 2004年07月 [査読有り][通常論文]
     
    Stearoyl-acyl carrier protein Delta(9) desaturase (Delta9D) produces oleic acid, a nutritionally valuable fatty acid containing a cis double bond between C-9 and C-10. This multiprotein diiron enzyme complex reacts with stearoyl-acyl carrier protein, reduced [2Fe-2S] ferredoxin, and O(2) to complete the highly regiospecific and stereoselective desaturation reaction. Interactions with the acyl chain provide stability to the enzyme-substrate complex, give an energetic contribution to catalytic selectivity, and help to order the electron transfer, O(2) binding, and C-H bond cleavage steps of catalysis. Reactions with natural acyl chains indicate the involvement of a highly reactive diiron intermediate capable of oxidizing secondary C-H bonds (bond dissociation energy approximate to 95 kcal/mol), but also capable of diagnostic O-atom transfer reactions with the appropriate substrate analogues. For soluble Delta9D, the natural reaction may initiate at the C-10 position, in contrast to the well-established initial reactivity of the membrane enzyme homologue stearoyl-coenzyme A (CoA) Delta(9) desaturase at the C-9 position.
  • AM Orville, L Manning, DS Blehert, JM Studts, BG Fox, GH Chambliss
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 60 Pt 5 957 - 961 2004年05月 [査読有り][通常論文]
     
    Diffraction-quality crystals have been obtained of the xenobiotic reductase A (XenA) from Pseudomonas II-B, which was originally cultured from the contaminated soil of a World War II era munitions-manufacturing plant. Several complete X-ray diffraction data sets have been collected and analyzed. The native XenA data set includes reflections between 35 and 1.65 Angstrom. Four-wavelength MAD data sets from selenomethionine-enriched XenA and from three different ligand complexes are also reported. The XenA crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 84, b = 158, c = 57 Angstrom. Experimental phasing from analysis of the MAD data from selenomethionine-enriched XenA reveals the presence of two molecules in the asymmetric unit. They are related by a noncrystallographic 21 screw axis nearly parallel to the c axis, but offset by a quarter unit-cell translation. Thus, the local symmetry produces approximate systematic absences along the (001) principal axis and complicates the space-group determination.
  • Zhao Q, Frederick R, Seder K, Thao S, Sreenath H, Peterson F, Volkman BF, Markley JL, Fox BG
    Journal of structural and functional genomics 5 1-2 87 - 93 2004年 [査読有り][通常論文]
  • Thao S, Zhao Q, Kimball T, Steffen E, Blommel PG, Riters M, Newman CS, Fox BG, Wrobel RL
    Journal of structural and functional genomics 5 4 267 - 276 2004年 [査読有り][通常論文]
  • RD White, BG Fox
    BIOCHEMISTRY 42 25 7828 - 7835 2003年07月 [査読有り][通常論文]
     
    The fatty acid analogues 9- and 10-thiastearate were converted to acyl-ACP derivatives by in vitro enzymatic synthesis and reacted with the reconstituted soluble stearoyl-ACP Delta9 desaturase complex. Electrospray ionization mass spectral analysis of the acyl chains purified from the reaction mixtures showed that 10-thiastearoyl-ACP was converted to the 10-sulfoxide as the sole product. In the presence of 1802, the sulfoxide oxygen was found to be derived exclusively from O-2. This result confirms the ability of the soluble stearoyl-ACP desaturase to catalyze O atom transfer in the presence of the appropriate substrate analogue. Inhibition studies showed that 10-thiastearoyl-ACP was a mixed-type inhibitor of 18:0-ACP, with an apparent K-I of similar to10 muM. Comparable reactions of the stearoyl-ACP desaturase complex with 9-thiastearoyl-ACP gave the 9-sulfoxide as similar to5% of the total products, with the 0 atom again exclusively derived from O-2. The remaining 95% of the total products arose from an acyl chain cleavage reaction between S-9 and C-10. Matrix-assisted laser desorption ionization time-of-flight mass spectral analysis showed that 9-thiastearoyl-ACP had a mass of 9443 amu while the acyl chain cleavage product had a mass of 9322 amu, corresponding to the calculated mass of 8-mercaptooctanoyl-ACP. Recovery of the acyl chain from the ACP product gave the disulfide of 8-mercaptooctanoate (mass of 349.1 amu), arising from the dimerization of 8-mercaptooctanoate during product workup. Gas chromatography-mass spectral analysis also showed the accumulation of nonanal in sealed reaction vials, accounting for the other product of the acyl chain cleavage reaction. The reactivity at both the 9 and 10 positions of the thia-substituted acyl-ACPs is consistent with the proximity of both positions to the diiron center oxidant in the enzyme-substrate complex. Moreover, the differential reactivity of the 9- and 10-thiastearoyl-ACPs also suggests position-dependent consequences of the reaction within the Delta9D active site. Mechanisms accounting for both sulfoxidation and acyl cleavage reactions by the stearoyl-ACP Delta9 desaturase are proposed.
  • KS Lyle, JA Haas, BG Fox
    BIOCHEMISTRY 42 19 5857 - 5866 2003年05月 [査読有り][通常論文]
     
    Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O-2-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (-95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O-2 and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for similar to90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over similar to1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) similar to 1.5) and k(linear) ((D)k(linear) similar to 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.
  • KH Mitchell, CE Rogge, T Gierahn, BG Fox
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 7 3784 - 3789 2003年04月 [査読有り][通常論文]
     
    The present studies address the mechanism of aromatic hydroxylation used by the natural and G103L isoforms of the diiron enzyme toluene 4-monooxygenase. These isoforms have comparable catalytic parameters but distinct regiospecificities for toluene hydroxylation. Hydroxylation of ring-deuterated p-xylene by the natural isoform revealed a substantial inverse isotope effect of 0.735, indicating a change in hybridization from sp(2) to sp(3) for hydroxylation at a carbon atom bearing the deuteron. During the hydroxylation of 4-H-2(1)- and 3,5-H-2(2)-toluene, similar magnitudes of intramolecular isotope effects and patterns of deuterium retention were observed from both isoforms studied, indicating that the active-site mutation affected substrate orientation but did not influence the mechanism of hydroxylation. The results with deuterated toluenes show inverse intramolecular isotope effects for hydroxylation at the position of deuteration, normal secondary isotope effects for hydroxylation adjacent to the position of deuteration, near-quantitative deuterium retention in m-cresol obtained from 4-H-2(1)-toluene, and partial loss of deuterium from all phenolic products obtained from 3,5-H-2(2)-toluene. This combination of results suggests that an active site-directed opening of position-specific transient epoxide intermediates may contribute to the chemical mechanism and the high degree of regiospecificity observed for aromatic hydroxylation in this evolutionarily specialized diiron enzyme.
  • AM Orville, JM Studts, GT Lountos, KH Mitchell, BG Fox
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 59 Pt 3 572 - 575 2003年03月 [査読有り][通常論文]
     
    Single crystals have been obtained of the toluene 4-monooxygenase catalytic effector protein, the SeMet-enriched protein and a truncated isoform missing ten amino acids from the N-terminus. Complete X-ray diffraction data sets have been collected and analyzed to 2.0, 3.0 and 1.96 Angstrom resolution for the native, SeMet and truncated isoform crystals, respectively. The native and SeMet proteins crystallized in space group P6(1)22 (unit-cell parameters a = b = 86.41 +/- 0.15, c = 143.90 +/- 0.27 Angstrom), whereas the truncated isoform crystallized in space group P2(1)3 (a = b = c = 86.70 +/- 0.47 Angstrom). Matthews coefficient calculations suggest either two or three molecules per asymmetric unit in the P6(1)22 space group and two molecules per asymmetric unit in the P2(1)3 space group. Experimental phases from MAD analysis of the SeMet isoform and molecular replacement of the truncated isoform confirm the presence of two molecules per asymmetric unit in each case. These crystallographic results are the first available for the evolutionarily related but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family.
  • FAJ Rotsaert, JD Pikus, BG Fox, JL Markley, J Sanders-Loehr
    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 8 3 318 - 326 2003年02月 [査読有り][通常論文]
     
    The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe2S2 ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe2S2 ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform N-15-labeling of Anabaena Fe2S2 ferredoxin results in shifts of -1 cm(-1) in the Fe-S-t stretching modes at 282, 340, and 357 cm(-1). The N-15 dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No N-15 dependence occurs for the nu(Fe-S-b) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-mono-oxygenase system of Pseudomonas mendocina. Upon selective N-15-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S-t) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with N-15-histidine (N-15 in either the alpha1, delta1 or delta1, epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe2S2 and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.
  • FE Gomez, DE Bauman, JM Ntambi, BG Fox
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 2 316 - 326 2003年01月 [査読有り][通常論文]
     
    The effects of sterculic acid on cell size. adiposity, and tatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100muM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MIDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells Cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shoes that de novo synthesis and elongation of fatty acids. as well as cell differentiation. were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells. the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation. scd gene expression or SCD protein translation. (C) 2002 Elsevier Science (USA). All rights reserved.
  • JA Haas, BG Fox
    BIOCHEMISTRY 41 49 14472 - 14481 2002年12月 [査読有り][通常論文]
     
    Stearoyl-acyl carrier protein Delta(9)-desaturase (Delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced Delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting Delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) Delta9D. An similar to4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting Delta9D was estimated to have K(D2) approximate to 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-1)) relative to 18:0-ACP (1 s(-1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.
  • CE Rogge, BG Fox
    BIOCHEMISTRY 41 31 10141 - 10148 2002年08月 [査読有り][通常論文]
     
    Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O-2-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K-M values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to similar to3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.
  • FE Gomez, M Miyazaki, YC Kim, P Marwah, HA Lardy, JM Ntambi, BG Fox
    BIOCHEMISTRY 41 17 5473 - 5482 2002年04月 [査読有り][通常論文]
     
    The effects of dehydroepiandrosterone (DHEA) and 7-oxo-DHEA on the cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 preadipocyte cells are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell sorting shows that preadipocyte cells treated with methylisobutylxanthine, dexamethasone, and insulin (MDT) plus DHEA comprise a population distribution of predominantly large cells with reduced adiposity. In contrast, cells treated with MDT plus 7-oxo-DHEA comprise a population distribution of almost equal proportions of small and large cells that have an adiposity equivalent to cells differentiated with MDT alone. The cells treated with MDT plus DHEA have significantly reduced levels of total fatty acid, mainly due to a dramatic reduction in the level of palmitoleic (Delta(9)-16:1) acid. The cells treated with MDT plus 7-oxo-DHEA have a significantly increased level of total fat, primarily due to increased levels of Delta(9)-16:1 and palmitic (16:0) acids. At the molecular level, the DHEA-treated cells contain lowered amounts of SCD1 mRNA and antibody-detectable desaturase protein, while 7-oxo-DHEA-treated cells contained elevated levels of SCD1 mRNA and protein. Inhibition of differentiation in DHEA-treated cells was also suggested by a reduction in the mRNA level of the adipogenic gene aP2. At the level of microsomal enzymatic activity, SCD activity was decreased in DHEA-treated cells while the SCD activity was increased in 7-oxo-DHEA-treated cells. The changes in mRNA levels and enzyme activity were concentration-dependent and appeared as early as day 3 of the differentiation protocol. The results show that DHEA and 7-oxo-DHEA have distinct modes of action with respect to the complex transcriptional cascade required for differentiation. Furthermore, differences in the insulin-stimulated uptake of 2-deoxyglucose and in the activity of carnitine palmitoyl transferase observed from either DHEA- or 7-oxo-DHEA-treated cells support the ability of DHEA to produce a thermogenic effect in differentiating preadipocytes, while 7-oxo-DHEA promotes differentiation without other changes typical of thermogenesis.
  • KH Mitchell, JM Studts, BG Fox
    BIOCHEMISTRY 41 9 3176 - 3188 2002年03月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD) concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing > 96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an similar to20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents similar to60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absense or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V-max/K-M specificities differing by similar to2-4-fold and coupling efficiencies ran,,in,, from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.
  • W Luo, LA Moe, L Skjeldal, JD Pikus, JL Markley, BG Fox
    JOURNAL OF BIOMOLECULAR NMR 21 1 73 - 74 2001年09月 [査読有り][通常論文]
  • Popescu VC, Münck E, Fox BG, Sanakis Y, Cummings JG, Turner IM Jr, Nelson MJ
    Biochemistry 40 27 7984 - 7991 2001年07月 [査読有り][通常論文]
     
    The alpha beta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH-. The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NHdark The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NHdark to a low-spin ferric complex, NHlight exhibiting an S = 1/2 EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NHdark and NHlight With Mossbauer spectroscopy. The z-axis of the Fe-57 magnetic hyperfine tenser, A, of NHlight was found to be rotated by similar to 45 degrees relative to the z-axis of the g tenser (g(z) = 1.97). Comparison of the A tenser of NHlight With the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mossbauer spectroscopy. Photolysis at 4.2 K in the Mossbauer spectrometer yielded a five-coordinate low-spin ferric species, NHA which converted back into NHdark when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.
  • H Hemmi, JM Studts, YK Chae, JK Song, JL Markley, BG Fox
    BIOCHEMISTRY 40 12 3512 - 3524 2001年03月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O-2-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alpha beta gamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D H-1, C-13, and N-15 NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 Angstrom for the backbone and 1.24 Angstrom for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha -helices and seven beta -strands arranged in an N-terminal beta alpha beta beta and a C-terminal beta alpha alpha beta beta beta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta -sheets and the interactions between the alpha -helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M, trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase), The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and SI (29% sequence identity) did not. The H-1 NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.
  • SF Oppenheim, JM Studts, BG Fox, JS Dordick
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 90 3 187 - 197 2001年03月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase is a four-protein component diiron enzyme complex. The enzyme catalyzes the hydroxylation of toluene to give p-cresol with similar to 96% regioselectivity. The performance of the enzyme in two-phase reaction systems consisting of toluene, hexane, or perfluorohexane and an aqueous buffer was rested. In each of the cosolvent systems, containing up to 93% (v/v) of solvent, the enzyme was active and exhibited regioselectivity indistinguishable from the aqueous reaction. Using the perfluorohexane/buffer system, a number of polycyclic aromatic hydrocarbons were oxidized that were not readily oxidized in aqueous buffer. An instability of the hydroxylase component and a substantial uncoupling of NADH utilization and product formation were observed in reactions that were continued for longer than similar to3 min. More stable enzyme complexes will be needed for broad applicability of this hydroxylating system in nonaqueous media.
  • JA Haas, MA Frederick, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 20 2 274 - 284 2000年11月 [査読有り][通常論文]
     
    Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label, Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall similar to 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K-M = 2.7 muM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta (9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta (9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported. (C) 2000 Academic Press.
  • JW Pak, KL Knoke, DR Noguera, BG Fox, GH Chambliss
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 66 11 4742 - 4750 2000年11月 [査読有り][通常論文]
     
    The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-(14)C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2,6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic mit of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of similar to0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O(2), but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP(+), Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The mit 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the mit 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl, A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.
  • JM Studts, KH Mitchell, JD Pikus, K McClay, RJ Steffan, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 20 1 58 - 65 2000年10月 [査読有り][通常論文]
     
    Toluene 4-monooxygenase is a four-protein complex that catalyzes the O-2- and NADH-dependent oxidation of toluene to p-cresol, The influence of various expression systems on the host cell growth characteristics, purified protein yields, and specific activity of the hydroxylase (T4moH) component of the complex was evaluated by considering the cell mass obtained per liter of fermentation culture medium, the purified protein obtained per gram of cell mass, and the specific activity of purified T4moH, The specific activity of purified T4moH was determined to be 1200-1250 nmol of p-cresol formed per minute per milligram of T4moH in air-saturated 50 mM phosphate buffer, pH 7.5, at 25 degrees C in the presence of optimal concentrations of the other protein components of the complex, saturating toluene (5.8 mM at 25 degrees C), and saturating NADH (1 mM), This value was obtained for T4moH purified from several different expression systems and apparently represents the maximal specific activity of the enzyme complex for toluene hydroxylation. By manipulation of vectors and gene inserts to eliminate adventitious catalytic turnover of NADH, up to 60-fold increase in the volumetric yield of T4moH activity was obtained from recombinant fermentations in Escherichia coli BL21(DE3). (C) 2000 Academic Press.
  • YC Kim, FE Gomez, BG Fox, JM Ntambi
    JOURNAL OF LIPID RESEARCH 41 8 1310 - 1316 2000年08月 [査読有り][通常論文]
     
    Two stearoyl-CoA desaturase (SCD) isoforms can be expressed during the differentiation of 3T3-L1 preadipocytes into adipocytes, Here we report on the effects of the peroxisome proliferator-activated receptor gamma ligand troglitazone (TRO) on scd1 and scd2 mRNA levels as determined by Northern blotting, on SCD protein expression as determined by Western blotting, and on total lipid composition as determined by GC during differentiation. In preadipocytes, scd1 mRNA and SCD protein were not detected, whereas scd2 mRNA was detected. These cells have high levels of palmitate (16:0), stearate (18:0), and monounsaturated oleate (Delta(9)-18:1) and low levels of monounsaturated palmitoleate (Delta(9)-16:1). In MDI (methylisobutylxanthine, dexamethasone, and insulin)-treated cells, scd1 mRNA and SCD protein were increased similar to 100-fold relative to preadipocyte levels, the scd2 mRNA level was increased 2-fold, Delta(9)-16:1 was increased similar to 20-fold, and 18:0 was decreased similar to 3-fold, In TRO-treated cells, the scd1 mRNA level was lower than that observed in preadipocytes, while the scd2 mRNA level was similar. TRO also decreased scd1 mRNA in primary adipocytes, The TRO-treated cells contained a Delta(9)-18:1 level typical of MDI-treated cells whereas, conversely, these cells also contained a low Delta(9)-16:1 level typical of preadipocytes. The implications of these correlations for the regulatory and enzymatic mechanism(s) used to establish and maintain lipid composition are discussed.
  • KS Lyle, P Moenne-Loccoz, JY Ai, J Sanders-Loehr, TM Loehr, BG Fox
    BIOCHEMISTRY 39 34 10507 - 10513 2000年08月 [査読有り][通常論文]
     
    Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of v(s)(Fe-O-Fe) and v(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from similar to 123 degrees in the ore-bridged diferric centers of the as-isolated enzyme to similar to 120 degrees in ore-bridged diferric centers of the complex. Analysis of the shifts in v(s)(Fe-O-Fe) and v(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta 9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta 9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta 9D was used to investigate whether an O-atom from O-2 would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of similar to 70% yield of 18:1-ACP product in the presence of O-18(2), no incorporation of an O-18 atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O-2 into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta 9D catalysis are discussed.
  • H Toyoda, A Kinoshita-Toyoda, B Fox, SB Selleck
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 29 21856 - 21861 2000年07月 [査読有り][通常論文]
     
    Mutations that disrupt developmental patterning in Drosophila have provided considerable information about growth factor signaling mechanisms. Three genes recently demonstrated to affect signaling by members of the Wnt, transforming growth factor-beta, Hedgehog, and fibroblast growth factor families in Drosophila encode proteins with homology to vertebrate enzymes involved in glycosaminoglycan synthesis. We report here the biochemical characterization of glycosaminoglycans in Drosophila bearing mutations in sugarless, sulfateless, and tout-velu. We find that mutations in sugarless, which encodes a protein with homology to UDP-glucose dehydrogenase, compromise the synthesis of both chondroitin and heparan sulfate, as would be predicted from a defect in UDP-glucuronate production. Defects in sulfateless, a gene encoding a protein with similarity to vertebrate N-deacetylase/N-sulfotransferases, do not affect chondroitin sulfate levels or composition but dramatically alter the composition of heparin lyase-released disaccharides. N-, 6-O-, and a O-sulfated disaccharides are absent and replaced entirely with an unsulfated disaccharide, A mutation in tout-velu, a gene related to the vertebrate Exostoses 1 heparan sulfate co-polymerase, likewise does not affect chondroitin sulfate synthesis but reduces all forms of heparan sulfate to below the limit of detection. These findings show that sugarless, sulfateless, and tout-velu affect glycosaminoglycan biosynthesis and demonstrate the utility of Dro sophila as a model organism for studying the function and biosynthesis of glycosaminoglycans in vivo.
  • K McClay, BG Fox, RJ Steffan
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 66 5 1877 - 1882 2000年05月 [査読有り][通常論文]
     
    Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomanas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 033 mu mol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.
  • H Hemmi, JM Studts, YK Chae, JL Markley, BG Fox
    JOURNAL OF BIOMOLECULAR NMR 16 4 359 - 360 2000年04月 [査読有り][通常論文]
  • JD Pikus, KH Mitchell, JM Studts, K McClay, RJ Steffan, BG Fox
    BIOCHEMISTRY 39 4 791 - 799 2000年02月 [査読有り][通常論文]
     
    The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O-2-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallo-graphically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O-2 activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-1) respectively, and k(cat)/K-M values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased similar to 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2,5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.
  • JA Broadwater, BJ Laundre, BG Fox
    JOURNAL OF INORGANIC BIOCHEMISTRY 78 1 7 - 14 2000年01月 [査読有り][通常論文]
     
    Positional isomers of mono-unsaturated 18:1-ACP have been used as substrates for stearoyl-acyl carrier protein Delta(9) desaturase to test whether a C-H bond abstraction from either the C-9 or C-10 position could lead to rearranged products diagnostic for the production of an allylic radical intermediate. The reconstituted enzyme complex was able to desaturate trans-Delta(11)-18:1-ACP and trans-Delta(7)-18:1-ACP, but not trans-Delta(9)-18:1-ACP, or any of the corresponding cis-isomers. Enzymatic desaturation of trans-Delta(11)-18:1-ACP gave a single product, cis-Delta(9),trans-Delta(11)-18:2-ACP, as characterized by gas chromatography-electron ionization mass spectrometry of the molecular ions, the fragmentation products of pyrrolidide and 4,4-dimethyloxazoline derivatives, and by comparison of chromatographic retention times with authentic standards. Reaction of trans-Delta(7)-18:1-ACP gave two enzymic products, trans-Delta(7),cis-Delta(9)-18:2 ( similar to 80%) and trans-Delta(7),cis-Delta(10)-18:2 ( similar to 20%). The major product was likely formed in a reaction identical to that of 18:0-ACP desaturation, while the minor product was likely formed by alternative placement of the C-10 and C-11 positions of the substrate analog in a cis configuration relative to the diiron oxidant. Since none of the products observed are indicative of rearrangements originating with an allylic radical, a discussion of the origins and possible implications of these results is presented. (C) 2000 Elsevier Science Inc. All rights reserved.
  • DS Blehert, BG Fox, GH Chambliss
    JOURNAL OF BACTERIOLOGY 181 20 6254 - 6263 1999年10月 [査読有り][通常論文]
     
    The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyelohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5 alpha. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.
  • Broadwater JA, Achim C, Münck E, Fox BG
    Biochemistry 38 38 12197 - 12204 1999年09月 [査読有り][通常論文]
     
    Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:O-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O-2 yields a blue chromophore (lambda(max) similar to 700 nm) that exhibits a vibrational spectrum indicative of a mu-1,2-peroxo complex; this species has been designated peroxo Delta 9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) similar to 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H2O2 or product (18:1-ACP). In this work, optical, transient kinetic, and Mossbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O-2-equilibrated 18:O-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-1) at 6 degrees C). The Mossbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E-Q(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E-Q(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E-Q(1) = 1.91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E-Q(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:O-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the Mossbauer cryostat caused the release of O-2 and the reappearance of a diferrous Delta 9D . 18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the Mossbauer spectrum of peroxo Delta 9D, showing that the released O-2 was effectively sequestered within the active site.
  • JA Haas, BG Fox
    BIOCHEMISTRY 38 39 12833 - 12840 1999年09月 [査読有り][通常論文]
     
    Stearoyl acyl carrier protein Delta(9) desaturase (Delta 9D) uses a diiron center to catalyze the NADPH- and O-2-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta 9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O-2 consumption and olefin production was found to be maximal for 18: 0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the KM values were similar (similar to 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by similar to 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K-M) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K-M values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The Delta Delta G(binding) estimated from the change in k(cat)/K-M for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R., and Tanford, C. (1973) PI-oc. Natl. Acad. Sci. U.S.A. 70, 289-293], Since the KM values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta 9D.
  • JM Studts, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 16 1 109 - 119 1999年06月 [査読有り][通常論文]
     
    An increasing demand for isotopically labeled samples for spectroscopic and crystallographic studies has led to a corresponding need for effective and efficient methods for producing these samples. The present work is based on the strategy of using an isotopically labeled compound as the growth-limiting nutrient during protein expression in Escherichia coli (DE3) strains. By using dissolved O-2 and agitation rate data, the cell growth, feeding of the isotopic label, induction of protein expression, and the harvest of cells can be coordinated in a feedback controlled fermenter in a simple, easily defined manner. This approach is demonstrated for the nutrient-limited production of [U-N-15]- and [U-C-13, U-N-15]-labeled toluene 4-monooxygenase effector protein in E. coli BL21(DE3) with isotopic abundance identical to that of the labeled precursors. For selective labeling, demonstrated with selenomethionine using methionine auxotroph E. coli B834(DE3), similar to 80-85% incorporation was obtained from methionine-dependent growth of the auxotroph followed by selenomethionine feeding and protein induction upon methionine depletion. This selective labeling is accomplished in a single culture, does not require washing or resuspension, minimizes costly incorporation of label into host cell mass prior to induction, and can be easily adapted to selective labeling with other amino acids. Moreover, cell mass yield from these experiments can be readily optimized to provide the desired level of protein for a given investigation from a single growth and purification. This combination provides an efficient, controllable option for isotopic labeling experiments. (C) 1999 Academic Press.
  • JA Broadwater, BG Fox
    PROTEIN EXPRESSION AND PURIFICATION 15 3 314 - 326 1999年04月 [査読有り][通常論文]
     
    Spinach ACP isoform I was overexpressed in Escherichia coli BL21(DE3) using a gene synthesized from codons associated with high-level expression in E. coli The synthetic gene has extensive changes in codon usage (23 of 77 total codons) relative to that of the originally synthesized plant gene (P. D. Beremand et at, 1987, Arch. Biochem Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His(6) were obtained in yields of up to 70 mg L-1 of culture medium, compared to similar to 1-6 mg L-1 of purified ACP obtained from the gene composed of predicted spinach codons. In either shaken flask or fermentation culture, similar to 15% conversion to holo-ACP or holo-ACP-Ris, was obtained regardless of the level of protein expression. However, coexpression of ACP-His(6) with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O-2-controlled fermentation routinely yielded greater than 95% conversion to holo-ACP-Hiss. Electrospray ionization mass spectrometric analysis of the purified recombinant ACPs revealed that the amino terminal. Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. This observation is consistent with the inhibition of endogenous Met-aminopeptidase by removal of catalytically essential Co(II) and introduces the importance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-His(6) was shown to be indistinguishable from stearoyl-ACP as a substrate for enzymatic acylation and desaturation, In combination, these studies provide a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies. (C) 1999 Academic Press.
  • YS Yang, JA Broadwater, SC Pulver, BG Fox, EI Solomon
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 121 12 2770 - 2783 1999年03月 [査読有り][通常論文]
     
    Stearoyl-acyl carrier protein (stearoyl-ACP) Delta(9)-desaturase (Delta(9)D) catalyzes the insertion of a cis double bond between the 9 and 10 positions of the stearoyl-ACP to convert it to oleoyl-ACP. The binuclear non-heme iron active site of the fully reduced enzyme (reduced Delta(9)D) and its substrate-bound form (stearoyl-ACP Delta(9)D) have been studied using a combination of circular dichroism (CD) and magnetic circular dichroism (MCD) to probe their geometric and electronic structures. CD and MCD in the near-IR region probe the ligand-field d-d transitions of the ferrous sites. Variable-temperature variable-field (VTVH) MCD combined with a spin-Hamiltonian analysis including the zero-field splitting (ZFS) of both irons and the exchange coupling (J) between the irons due to bridging ligation is used to probe their ground-state properties. These ground- and excited-state results indicate that the active site of reduced Delta(9)D has two equivalent 5-coordinate irons in a distorted square pyramidal geometry. They are weakly antiferromagnetically coupled with large negative and equivalent ZFSs (D-1 = D-2 < 0), which gives a diamagnetic ground state interacting with low-lying paramagnetic excited states. Addition of substrate causes a significant change in both the excited states and the nature of the ground state. These spectral changes indicate that one of the irons becomes it-coordinate, while the other distorts toward a trigonal bipyramidal geometry. The two irons remain weakly antiferromagnetically coupled. However, this geometry change modifies their ZFSs (D-1 < 0 and D-2 > 0), which results in a new ground state, M-s = +/-1, with a low-lying M-s = +/-2 first excited state. These results are the first direct evidence that the stearoyl-ACP binding strongly perturbs the active site, creating an additional open coordination position that correlates with enhanced dioxygen reactivity. These results are correlated with the X-ray crystal structure and compared to the related enzyme, ribonucleotide reductase, to,gain insight into geometric and electronic structure contributions to dioxygen reactivity.
  • JA Broadwater, JA Haas, C Achim, E Munck, JY Ai, J Sanders-Loehr, TM Loehr, BG Fox
    ENZYMATIC MECHANISMS 27 162 - 175 1999年 [査読有り][通常論文]
     
    The diiron enzyme stearoyl-acyl carrier protein (ACP) Delta(9) desaturase catalyzes the NAD(P)H- and O-2-dependent desaturation of 18:0-ACP to give 18:1-ACP. The reconstituted catalytic system is a four protein complex consisting of ferredoxin reductase, [2Fe-2S] ferredoxin, the terminal homodimeric desaturase, and the fourth protein component, which is the substrate, 18:0-ACP. Genetic engineering, coexpression and fermentation have been required to produce sufficient quantities of recombinant acyl-ACPs for expanded studies of enzymatic desaturation. Among the well-characterized diiron enzymes, we have found that diferrous Delta 9D is distinguished by an extremely slow rate of single turnover autooxidation, yet reacts with 18:0-ACP under steady stare conditions to give 18:1-ACP with >10(3) increase in oxidation rate. Moreover, previous studies indicated that the desaturase exhibits similar to 10(2) fold higher V-max for 18:0-ACP as compared to 16:0-ACP, while the apparent K-m-values were indistinguishable. These studies suggest the importance of the acyl portion of the substrate in activating the enzyme for catalysis. This suggestion is substantiated by our recent studies of complexes of diferrous enzyme, 18:0-ACP, and O-2. Optical and resonance Raman studies revealed an intermediate with absorption at similar to 700 nm, an O-O symmetric vibration at 898 cm(-1), and relatively long lifetime (t(1/2) approximate to 23 min). These spectral characteristics suggest a quasi-stable peroxy differic intermediate. Mossbauer studies revealed an antiferromagnetically coupled, symmetric intermediate with an isomer shift of similar to 0.85 mm/s; this intermediate value of isomer shift has not been observed in any previous studies of diiron enzymes. Under single turnover conditions, the complex decays to the resting state without production of 18:I-ACP. However, since no detectable levels of H2O2 were obtained from this reaction, an oxidase reaction arising from reaction at one of the two available diiron centers is proposed. By coupling formation of a relatively stable peroxy diferric intermediate with subsequent electron and proton transfers, this reaction provides a catalytic precedent for the "alternative oxidase" activity recently assigned to a membrane diiron enzyme in thermogenic plants and trypanosomes.
  • B Xia, JD Pikus, WD Xia, K McClay, RJ Steffan, YK Chae, WM Westler, JL Markley, BG Fox
    BIOCHEMISTRY 38 2 727 - 739 1999年01月 [査読有り][通常論文]
     
    T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary H-1 NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted H-1 resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted H-1, H-2, and N-15 signals from T4MOC. Selective deuteration led to identification of signals from the four Hii atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the HE protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the H-1 spectrum of reduced T4MOC was assigned on the basis of selective H-2 labeling to the His H-epsilon 1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond H-1-C-13 correlation (at 25.39 ppm H-1 and 136.11 ppm C-13) observed in spectra of [U-C-13]T4MOC with a H-1-C-13 coupling constant of similar to 192 Hz, The carbon chemical shift and one bond coupling constant are those expected for H-1(epsilon 1)-C-13(epsilon 1) in the imidazolium ring of histidine and are inconsistent with values expected for cysteine H-1(alpha)-C-13(alpha). The His H-epsilon 1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A H-1 peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain H-1 assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective N-15 labeling was used to identify hyperfine-shifted N-15 NMR signals from the backbone nitrogens of ail four cluster ligands (C45, H47, C64, and H67), from the N-epsilon 2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the N-delta 1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bunds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (150 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.
  • JA Broadwater, JY Ai, TM Loehr, J Sanders-Loehr, BG Fox
    BIOCHEMISTRY 37 42 14664 - 14671 1998年10月 [査読有り][通常論文]
     
    Combined optical and resonance Raman studies have revealed the formation of an Op-adduct upon exposure of 4e(-) chemically reduced stearoyl-acyl carrier protein Delta(9) desaturase to stearoyl-AGP and 1 atm O-2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t(1/2) approximate to 26 min). Resonance Raman studies using O-16(2) gas reveal vibrational features of a bound peroxide [v(s)(Fe-O-2), 442 cm(-1); v(as)(Fe-O-2), 490 cm(-1); v(O-O), 898 cm(-1)] that undergo the expected mass-dependent shifts when prepared in (OO)-O-16-O-18 or O-18(2.) The appearance of two Fe-O-2 vibrations, each having a single peak of intermediate frequency with (OO)-O-16-O-18, proves that the peroxide is bound symmetrically between the two iron atoms in a mu-1,2 configuration. The same results have been obtained in the accompanying resonance Raman study of ribonucleotide reductase isoform W48F/D84E [P. Moenne-Loccoz, J. Baldurin, B. A. Ley, T. M. Loehr, and J. M. Bollinger, Jr. (1998) Biochemistry 37, 14659-14663], thus making it likely that other members of the class II diiron enzymes form related peroxodiferric intermediates. Study of the reactivity of peroxodiferric Delta 9D revealed that this intermediate underwent 2e(-) reduction leading to an oxidase reaction and recovery of the resting ferric homodimer. In contrast, biological reduction of the same enzyme preparations using ferredoxin reductase and [2Fe-2S] ferredoxin gave catalytic desaturation with a turnover number of 20-30 min(-1). The profound difference in catalytic outcome for chemically and enzymatically reduced Delta 9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta 9D oxidase reaction represents a new type of reactivity for the acyl-ACP desaturases and provides a two-step catalytic precedent for the "alternative oxidase" activity recently proposed for a membrane diiron enzyme in plants and trypanosomes.
  • LJ Shu, JA Broadwater, C Achim, BG Fox, E Munck, L Que
    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 3 4 392 - 400 1998年08月 [査読有り][通常論文]
     
    Stearoyl-acyl carrier protein (ACP) Delta(9)-desaturase (Delta 9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O-2- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation slate. These studies re veal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41 Angstrom. The species having the 3.12 Angstrom Fe-Fe distance also exhibits a 1.8 Angstrom Fe-O bond and is thus proposed to represent Delta 9D molecules containing a (mu-oxo)bis(mu-carboxylato)diiron(III) cluster. The species having the 3.41 A Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Delta 9D molecules containing a (mu-hydroxo)diiron(III) cluster. Mossbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (Delta E-Q(1) =1.53 mm/s, 72%. Delta E-Q(2) = 0.72 mm/s, 21%; Delta E-Q(3) =2.20 mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (mu-oxo)- and (mu-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mossbauer majority species, is likely associated with the EXAFS majority species having a 3.12 Angstrom Fe-Fe separation and a 1.8 Angstrom Fe-mu-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41 Angstrom Fe-Fe separation. The presence of multiple diiron center conformations in diferric Delta 9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (similar to 9 kDa) during desaturation catalysis.
  • JA Broadwater, JA Haas, BG Fox
    FETT-LIPID 100 4-5 103 - 113 1998年05月 [査読有り][通常論文]
     
    Diiron enzymes catalyze many essential O-2-dependent reactions required for eukaryotic and bacterial metabolism Both membrane bound and soluble classes are now known. The integral membrane class of diiron enzymes contains desaturases, hydroxylases, and other oxidative enzymes involved in the synthesis of nutritionally or commercially desirable unsaturated fats, steroids, and other hydrophobic molecules. The soluble class of diiron enzymes has essential roles in DNA biosynthesis, desaturation of various acyl-ACPs, and the oxidation of hydrocarbons including toxic environmental pollutants. Biochemical, spectroscopic, and crystallographic studies of these enzymes have provided structures for the active sites and proposals for the mechanism of action. Two key features of the reaction cycles are the flexibility of the diiron coordination environment and the activation of O-2 to generate a series of related but not identical intermediates used for diverse catalytic processes. Most recently, the alpha helical bundle containing the diiron center has proven to be a robust scaffold for mutagenesis studies, allowing the relationship between protein structure and catalytic function to be further refined and adapted.
  • DS Blehert, KL Knoke, BG Fox, GH Chambliss
    JOURNAL OF BACTERIOLOGY 179 22 6912 - 6920 1997年11月 [査読有り][通常論文]
     
    Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C, While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a K-m for NG of 52 +/- 4 mu M, a K-m for NADPH of 28 +/- 2 mu M, and a V-max of 124 +/- 6 mu M . min(-1), while the P. fluorescens enzyme had a K-m for NG of 110 +/- 10 mu M, a K-m for NADPH of 5 +/- 1 mu M, and a V-max of 110 +/- 11 mu M . min(-1). Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an approximate to 2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions of middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.
  • JD Pikus, JM Studts, K McClay, RJ Steffan, BG Fox
    BIOCHEMISTRY 36 31 9283 - 9289 1997年08月 [査読有り][通常論文]
     
    Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. The diiron center is contained in the tmoA polypeptide of the hydroxylase component [(alpha beta gamma)(2), M-r approximate to 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for pam hydroxylation of toluene (k(cat), approximate to 2 s(-1) with respect to an alpha beta gamma protomer), o-xylene (k(cat) approximate to 0.8 s(-1)), m-xylene (k(cat) approximate to 0.6 s(-1)), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (k(cat) approximate to 0.4 s(-1)), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximate to 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The k(cat) of purified Q141C for toluene oxidation was approximate to 0.2 s(-1). Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relative to the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 and F205 are suggested to lie in a hydrophobic region closer to the Fe-A iron site, while I180 will be closer to Fe-B. These studies reveal that changes in the hydrophobic region predicted to be nearest to Fe-A can influence the regiospecificity observed for toluene 4-monooxygenase.
  • Shanklin J, Achim C, Schmidt H, Fox BG, Münck E
    Proceedings of the National Academy of Sciences of the United States of America 94 7 2981 - 2986 1997年04月 [査読有り][通常論文]
     
    The gene encoding the alkane omega-hydroxylase (AlkB; EC 1.14.15.3) from Pseudomonas oleovorans was expressed in Escherichia coli, The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins, Purified AlkB had specific activity of up to 5 units/mg for octane-dependent NADPH consumption, Mossbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) Delta(9) desaturase, rubrerythrin, and purple acid phosphatase, In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(lII) sites, with an occupancy of up to 0.9 cluster per AlkB, The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air, When both O-2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site, Mossbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands, New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase, Based on our Mossbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters, Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O-2-activation biochemistry.
  • JY Ai, JA Broadwater, TM Loehr, J SandersLoehr, BG Fox
    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 2 1 37 - 45 1997年02月 [査読有り][通常論文]
     
    The stearoyl-acyl carrier protein Delta(9) desaturase (Delta(9)D) uses an ore-bridged diiron center to cata lyze the NAD(P)H- and O-2-dependent desaturation of stearoyl-ACP. Delta 9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O-2 to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Delta 9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k(app) approximate to 3.5 s(-1) in 0.7 M NaN3, pH 7.8). RR experiments demonstrate the existence of two different adducts: an eta(1)-terminal structure at pH 7.8 (N-14(3)- asymmetric stretch at 2073 cm(-1), resolved into two bands with (NN2-)-N-15-N-14) and a mu-1,3 bridging structure at pH < 7 (N-14(3)- asymmetric stretch at 2100 cm(-1), shifted as a single band with (NN2-)-N-15-N-14). Both adducts also exhibit an Fe-N-3 stretching mode at approximate to 380 cm(-1), but no accompanying Fe-O-Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a mu-1,3 bridging azide supports the likelihood of a mu-1,2 bridging peroxide as a catalytic intermediate in the Delta 9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries.
  • K McClay, BG Fox, RJ Steffan
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 62 8 2716 - 2722 1996年08月 [査読有り][通常論文]
     
    Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp, strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein), Degradation of [C-14]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (similar to 30 to 57% of the total products), soluble metabolites (similar to 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (similar to 30%), and chloride ions (similar to 75% of the expected yield), CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1, Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.
  • Pikus JD, Studts JM, Achim C, Kauffmann KE, Münck E, Steffan RJ, McClay K, Fox BG
    Biochemistry 35 28 9106 - 9119 1996年07月 [査読有り][通常論文]
     
    Expression of the tmoA-F gene cluster from Pseudomonas mendocina KR1 in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity, These proteins are a diiron hydroxylase (T4MOH), a Rieske-type ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (alpha beta epsilon)(2), M(r) approximate to 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X((28-37))DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP Delta(9) desaturase. Mossbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mossbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX((15-17))CX(2)H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH, The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.
  • JL Markley, B Xia, YK Chae, H Cheng, WM Westler, JD Pikus, BG Fox
    PROTEIN STRUCTURE - FUNCTION RELATIONSHIP 135 - 146 1996年 [査読有り][通常論文]
  • BJ HOFFMAN, JA BROADWATER, P JOHNSON, J HARPER, BG FOX, WR KENEALY
    PROTEIN EXPRESSION AND PURIFICATION 6 5 646 - 654 1995年10月 [査読有り][通常論文]
     
    A method for producing recombinant proteins in pilot scale fermentation equipment using a glucose fed-batch initial growth, followed by a midlog phase feeding of a glucose and lactose mixture is described. Using the host strain Escherichia coli BL21(DE3), the diiron protein stearoyl-acyl carrier protein Delta(9) desaturase has been overexpressed at a biomass level of up to 12 g x liter(-1) dry cell weight, representing a 12-fold increase in volumetric productivity relative to that obtained from batch fermentations. Under these conditions, a maximum of 36% of the total cellular protein accumulates as the desaturase polypeptide. A correlation between the slowed growth rate of the fed-batch culture, a continued, albeit slower, exponential growth under inducing conditions, and a favorable partitioning between formation of the soluble holoprotein and inclusion bodies is reported. This correlation suggests that fed-batch techniques can be used to beneficially influence rate-limiting processes in the maturation of overexpressed proteins, such as metal uptake and incorporation proposed here. By using cells produced from the fed-batch method, the iron-containing, soluble desaturase can be purified in a yield of up to 66 mg x g(-1) dry cell weight (similar to 500 mg X liter(-1) culture), representing a three to fivefold increase in the yield relative to that obtained from batch fermentations. In addition, these methods are suitable for the production of the Anabena 7120 vegetative [2Fe 2S] ferredoxin in E. coli BL21(DE3) pLysS, a host strain used for the overexpression of toxic proteins. (C) 1995 Academic Press, Inc.
  • 藤井 浩, Yanhong Dong, Hiroshi Fujii, Michael P. Hendrich, Randolph A. Leising, Ganfeng Pan, Clyton R. Randall, Elizabeth C. Wilkinson, Yan Zang, Lawrence Que, Jr, Brian G. Fox, Karl Kauffmann, Eckard Münck
    J. Am. Chem. Soc. 117 10 2778 - 2792 1995年03月 [査読無し][通常論文]
     
    In our efforts to model the oxygen activation chemistry of methane monooxygenase (MMO) and the R2 protein of ribonucleotide reductase (RNR), we have discovered a transient green species (3) in the reaction of H2O2 with a (mu-oxo)diiron(III) TPA complex (TPA = tris(2-pyridylmethyl)amine). Our studies show that the precursor to 3 is [Fe2O(TPA)(2)(OH)(H2O)](ClO4)(3) (2a), which can be obtained by the treatment of [Fe2O(TPA)(2)(H2O)(ClO4)]-(ClO4)(3) (1) With an equivalent of base. Crystallographic studies show that 1 has a nearly linear (mu-oxo)diiron(III) core with terminal aqua and perchlorato ligands (angle Fe-(mu-O)-Fe = 174.1(4)degrees), while 2c, the 5-Et-TPA analogue of 2a, has a bent (mu-oxo)diiron(III) core that is supported by an H3O2- bridge, The presence of an H3O2- bridge in the latter is indicated by the short O-O separation (2.464(9) Angstrom), the Fe-Fe distance of 3.346(9) Angstrom, and the Fe-(mu-O)-Fe angle of 136.3(3)degrees. Thus treatment of 1 with an equivalent of base results in the replacement of the bound perchlorate with hydroxide and the bending of the Fe-O-Fe unit to form 2, That the bent Fe-O-Fe core persists in solution is indicated by its UV-vis features and NMR spectra that reflect distinct TPA coordination modes about the individual iron sites. The green intermediate 3 is generated by the reaction of 2, [Fe2O(L)(2)(OH)(H2O)](ClO4)(3) (L = TPA, 5-Me-TPA, and 5-Et-TPA), with H2O2 in CH3CN at -40 degrees C; when 5-Me-TPA is used as the tripodal ligand, 3b can be isolated as a solid upon standing overnight at -40 degrees C. Complex 3b exhibits electronic absorption features at 366 (epsilon = 7900 M(-1) cm(-1)) and 616 nm (epsilon = 5200 M(-1) cm(-1)) and an S = 3/2 EPR spectrum with g values at 4.45, 3.90, and 2.01. It exhibits one sharp Mossbauer doublet with Delta E(Q) = 0.49 mm/s and delta = 0.12 mm/s at 100 K, which accounts for 90% of the iron in the solid. Elemental analysis and electrospray ionization mass spectrometry show that 3b is a dinuclear complex best formulated as [Fe-2(O)(2)(5-Me-TPA)(2)](ClO4)(3). This dinuclear formulation is corroborated by magnetic susceptibility measurements showing that 3b has a high-temperature moment of 3.9 mu(B)/2Fe, corresponding to the 5 = 3/2 center observed by EPR. The formula for 3b suggests two unique properties: (a) that it has an Fe-2(mu-O)(2) core, and (b) that it is formally (FeFeIV)-Fe-III. The presence of an Fe-2(mu-O)(2) core in 3b is indicated by its EXAFS spectrum, which requires the inclusion of an Fe scatterer at 2.89 Angstrom for a satisfactory fit. It is further supported by the observation of resonance-enhanced Raman features at 676 and 656 cm(-1) (both of which shift to 634 cm(-1) with added (H2O)-O-18), which are associated with an Fe2O2 breathing mode by analogy to those observed for Mn2O2 complexes. The high-valent nature of 3b is corroborated by the ca. 3 eV upshift of its higher X-ray absorption K-edge relative to that of 2b and the reduction of 3b to the diiron(III) state at -40 degrees C by chemical (ferrocene titration) and cyclic voltammetric (E(1/2) = 0.96 V vs NHE) methods. Thus, 3b represents a bis(mu-oxo)-diiron complex with a formally (FeFeIV)-Fe-III valence state. Complex 3b has an unusual electronic structure. EPR, magnetization, and Mossbauer studies show that 3b has an S = 3/2 ground state with a large and nearly axial zero-field splitting, D = 35 +/- 15 cm(-1) and E/D = 0.04. The Mossbauer data show that 3 contains two equivalent iron sites which have unusually small magnetic hyperfine interactions, A = (-7.8, -7.9, -6.5) MHz. A variety of exchange coupling models are considered to describe the electronic properties of 3b; these include (FeFeIII)-Fe-III sites coupled to a ligand radical and valence-delocalized (FeFeIV)-Fe-III centers. Among the models considered, the only one that could possibly explain the observed site equivalence, isomer shift, and other properties consists of a valence-delocalized low-spin (S = 1/2) Fe-III-low-spin (S = 1) Fe-IV pair coupled by Heisenberg as well as double exchange; however, detailed theoretical studies of double exchange interactions involving low-spin iron sites are required before such an assignment can be made. Whatever its electronic structure, 3b is the only well-characterized high-valent nonheme iron species that is derived from the reaction of H2O2 and a (mu-oxo)diiron(III) complex. As such, it is relevant to the transient species observed in the oxidation chemistry of MMO and RNR R2, and provides a synthetic example of how a high-valent state can be attained in a nonheme environment.
  • BG FOX, J SHANKLIN, JY AI, TM LOEHR, J SANDERSLOEHR
    BIOCHEMISTRY 33 43 12776 - 12786 1994年11月 [査読有り][通常論文]
     
    The stearoyl-ACP Delta(9) desaturase from plants is a new example of a growing number of proteins that contain oxo- or hydroxo-bridged diiron clusters. On the basis of differences in primary sequence motifs providing the cluster ligands and upon structural differences elucidated by X-ray crystallography, we now propose that the presently known, soluble diiron-oxo proteins can be grouped into two classes, I and II. Class I contains hemerythrin, myohemerythrin, and, possibly, purple acid phosphatase. Class II contains ribonucleotide reductases, bacterial hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase, and phenol hydroxylase), rubrerythrin, and stearoyl-ACP desaturases. Through the use of resonance Raman spectroscopy, we have detected symmetric (v(s) = 519 cm(-1)) and asymmetric (v(as) = 747 cm(-1)) vibrational modes in the castor stearoyl-ACP Delta(9) desaturase, which are typical of ore-bridged diiron clusters. These frequencies shift by -18 and -34 cm(-1), respectively, in (H2O)-O-18, proving that the bridging ligand is readily exchangeable with solvent (t(1/2) = 7 min). Calculation of an similar to 123 degrees Fe-O-Fe angle from the position of v(s) and v(as) and from the O-18-dependent shift in these frequencies suggests that the diiron-oxo cluster in the desaturase is triply bridged in the diferric state. In the diferrous state, the two iron sites of the cluster are structurally inequivalent, as shown by differential temperature dependence of the Mossbauer quadrupole splittings. For the class II diiron-oxo proteins, primary sequence alignments reveal conserved amino acid residues which act as iron cluster ligands, participate in a hydrogen-bonding network, and are potentially involved in O-2 binding and activation. Based on this conservation, a structural model for the stearoyl-ACP Delta(9) desaturase active site is proposed that has strong similarity to both ribonucleotide reductase and methane monooxygenase. However, after single turnover of the diferrous state with O-18(2), O-18 is not detected in the oxo bridge of the castor desaturase. This is in contrast to the outcome observed for ribonucleotide reductase, suggesting the desaturase and ribonucleotide reductase differ in certain aspects of their respective O-2-activation reactions.
  • J SHANKLIN, E WHITTLE, BG FOX
    BIOCHEMISTRY 33 43 12787 - 12794 1994年11月 [査読有り][通常論文]
     
    The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O-2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains. Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 ?(or 4)) H,HX((2 or 3)) HH, and HX((2 or 3)) HH. This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase. Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice. The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains. Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin. In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat Delta(9) desaturase gene to complement a yeast strain deficient in the Delta(9) desaturase gene function (ole1). By site-directed mutagenesis, eight conserved His residues in the rat Delta(9) desaturase were individually converted to Ala. Each His --> Ala mutation failed to complement the yeast ole1 mutant. In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype. Western blot analysis showed that steady-state expression levels were equivalent for the wild-type rat desaturase and for all mutants, suggesting that the conserved His residues are essential for catalytic function. One role for these His residues would be to act as ligands for the iron atom(s) contained in these enzymes. On the basis of these results and the presently available spectroscopic data, we recognize the possibility that the membrane desaturases, alkane hydroxylase, and xylene monooxygenase contain a new structural type of diiron center.
  • J SHANKLIN, EJ WHITTLE, BG FOX
    PLANT LIPID METABOLISM 18 - 20 1994年 [査読有り][通常論文]
  • KE PAULSEN, Y LIU, BG FOX, JD LIPSCOMB, E MUNCK, MT STANKOVICH
    BIOCHEMISTRY 33 3 713 - 722 1994年01月 [査読有り][通常論文]
     
    Methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of hydroxylase (MMOH), reductase (MMOR), and ''B'' (MMOB) protein components. MMOH contains two oxygen-bridged dinuclear iron clusters that are the sites of O2 activation and hydrocarbon oxidation. Each cluster can be stabilized in diferric [Fe(III).Fe(III)], mixed-valence [Fe(II).Fe(III)], and diferrous [Fe(II).Fe(II)] redox states. We have correlated the EPR spin quantitation of the S = 1/2 mixed-valence state with the system electrode potential to determine both formal redox potential values for MMOH at 4-degrees-C: E1-degrees' = +76 +/- 15 mV and E2-degrees' = +21 +/- 15 mV (E(m) = +48 mV, 61% maximum mixed-valence state). Complementary Mossbauer studies of Fe-57-enriched MMOH allowed all three redox states to be quantitated simultaneously in individual samples and revealed that the distribution of redox states was in accord with the measured potential values. EPR spectra of partially reduced MMOH showed that the apparent midpoint potential values of MMOH-MMOR, MMOH-MMOR-MMOB, and MMOH-MMOR-MMOB-substrate complexes were slightly more positive than that of MMOH alone. In contrast, the MMOH-MMOB complex appeared to have a substantially more negative redox potential. The formal redox potential values of the latter complex were determined to be E1-degrees' = -52 +/- 15 mV and E2-degrees' = -115 +/- 15 mV, respectively, at 4-degrees-C (E(m) = -84 mV, 65% maximum mixed-valence state). This negative 132-mV shift in the midpoint potential of MMOH coupled to MMOB binding suggests that MMOB binds almost-equal-to 10(4) more strongly to the diferric state of MMOH than to the diferrous state. Since the potential shift is strongly negative, and since a nearly constant separation between the two formal potential values of MMOH is maintained when MMOB binds, the role of the MMOB-MMOH complex must not be to thermodynamically stabilize the formation of the diferrous cluster which is the form that reacts with O2 during catalysis. However, MMOB binding may provide kinetic stabilization of the diferrous state and/or modulation of the interaction of MMOH with O2 and hydrocarbon substrates. Such roles may be effected through cyclic association and dissociation of the MMOB-MMOH complex as MMOH oscillates between redox states during catalysis, thereby dynamically altering the affinity of this complex.
  • S PULVER, WA FROLAND, BG FOX, JD LIPSCOMB, EI SOLOMON
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 115 26 12409 - 12422 1993年12月 [査読有り][通常論文]
     
    A combination of circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies has been used to probe the geometric and electronic structure of the binuclear Fe(II) active site of the reduced hydroxylase component of methane monooxygenase (MMOH). Excited-state data provide the numbers and energies of d --> d transitions which are interpreted in terms of ligand field calculations to estimate the geometry of each iron. Variable-temperature variable-field (VTVH) MCD data are analyzed by using a non-Kramers doublet model to obtain the zero field splitting (ZFS) and g(parallel-to) value of the ground state and the excited sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe2+ combined with the exchange coupling between iron centers. The reduced MMOH contains two five-coordinate ferrous centers with different geometries. VTVH MCD data show the ferrous centers to be ferromagnetically coupled with J approximately 0.3-0.5 cm-1 for the reduced hydroxylase. This indicates that in contrast to deoxyHr which has a binuclear Fe2+ site that is antiferromagnetically coupled through a hydroxide bridge, fully reduced MMOH may have a water bridge. The addition of anions, substrates, and inhibitors to reduced MMOH results in no change in the CD spectrum suggesting that these molecules do not bind to the iron or cause large perturbations in the iron site. In contrast, addition of component B causes dramatic changes in the CD and MCD spectra which indicate that one iron in the biferrous active site is altered. Two ferromagnetically coupled Fe(II) centers with distorted five-coordinate square-pyramidal geometries are also found for the MMOH-component B complex. Geometric and electronic structural changes resulting from the addition of component B to reduced MMOH are described and correlated with enhanced reactivity. The above results are compared to parallel results for deoxyHr, and differences are correlated to the difference in dioxygen reactivity (binding versus activation).
  • KL KOSTKA, BG FOX, MP HENDRICH, TJ COLLINS, CEF RICKARD, LJ WRIGHT, E MUNCK
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 115 15 6746 - 6757 1993年07月 [査読有り][通常論文]
     
    Synthetic iron(IV) complexes are attracting interest both as models for putative intermediates of biochemical reactions as well as for proposed catalytic entities. We have previously reported the X-ray structure of [Et4N][Fe(IV)Cl(eta4-MAC)] (H-4[MAC] = 1,4,8,11-tetraaza-13,13-diethyl-2,2,5,5,7,7,10,10-octamethyl-3,6,9,12,14-pentaoxocy-clotetradecane), where iron is coordinated to a plane of four amide nitrogen anions of a macrocyclic ligand and to one axial chloride (Collins, T. J.; Kostka, K. L.; Munck, E.; Uffelman, E. J. Am. Chem. Soc. 1990,112, 5637-5639). In zero magnetic field, the 4.2 K Mossbauer spectrum of [Et4N] [FeIVCl(eta4-MAC)] was a single quadrupole doublet with DELTAE(Q) = 0.89 mm/s and delta = -0.02 mm/s, consistent with an iron(IV) assignment. Here we present full synthetic and characterization results together with detailed Mossbauer and integer spin EPR studies of [FeIVCl(eta4-MAC)]- prepared with [Ph4P]+, [Et4N]+, [n-Bu4N]+, and [PPN]+ counterions. In strong applied fields, the Mossbauer spectra exhibit magnetic hyperfine patterns typical of complexes with integer electronic spin. The zero-field splitting parameters (D = -2.6 cm-1 and E/D = 0. 1 3) are such that the two lowest spin levels of the ground multiplet are nearly degenerate (DELTA = 0.16 cm-1). Correspondingly, [FeIVCl(eta4-MAC)]- exhibits an integer spin EPR resonance at X-band with g(eff) = 8.0, indicative of a high-spin (S = 2) ground configuration. Quantitative analysis of the integer spin EPR spectra observed from both frozen CH3CN solution and from polycrystalline samples shows that the principal g-values are less than 2, as expected for high-spin iron(IV), and that the spin concentration of the S = 2 species agrees within 12% with the concentration determined by optical spectroscopy. We also report synthetic details and the X-ray structure of [Et4N]2[FeIIICl(eta4-MAC)].CH2Cl2.H2O. Crystals of [Et4N]2[FeIIICl(eta4-MAC)].CH2Cl2.H2O at 293 K are monoclinic, space group P2(1)/n, with a = 11.797(9) angstrom, b = 18.662(6) angstrom, c = 21.984(8) angstrom, beta = 102.75(6)degrees, V = 4708.6 angstrom3, Z = 4 (d(calcd) = 1.423 g cm-3), mu(alpha)(Mo Kalpha) = 3.92 cm-1, and R1 (unweighted, based on F) = 0.086 for 3298 observed reflections [I > 3sigma(I)]. Mossbauer and EPR studies show that this complex has an intermediate-spin (S = 3/2) ground state with hyperfine parameters similar to those reported for the porphyrin complex [FeIII(TPP)(FSbF5)] (Gupta, G. P.; Lang, G.; Reed, C. A.; Shelly, K.; Scheidt, W. R. J. Chem. Phys. 1987,86,5288-5293). However, the zero-field splitting tensor of [Et4N]2[FeIIICl(eta4-MAC)] (D = -3.7 cm-1, E/D = 0.05) is rotated by 90-degrees relative to the magnetic hyperfine and electric field gradient tensors. Analysis of the high-field Mossbauer spectra for both [FeIIICl(eta4-MAC)]2- and [FeIVCl(eta4-MAC)]- reveals magnetic hyperfine interactions substantially smaller than expected for an ionic complex, suggesting substantial covalency for both redox states.
  • BG FOX, MP HENDRICH, KK SURERUS, KK ANDERSSON, WA FROLAND, JD LIPSCOMB, E MUNCK
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 115 9 3688 - 3701 1993年05月 [査読有り][通常論文]
     
    Soluble methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of three components: hydroxylase, reductase, and component B. The active-site diiron cluster of the hydroxylase has been studied with Mossbauer, ENDOR, and EPR spectroscopies. Mossbauer spectra of the oxidized cluster show that the two high-spin irons are antiferromagnetically coupled in accord with our preliminary study (Fox et al. J. Biol. Chem. 1988, 263, 10553-10556). Mossbauer studies also reveal the presence of two cluster conformations at pH 9. The excited-state S = 2 multiplet of the exchange-coupled cluster (Fe3+.Fe3+) gives rise to an integer-spin EPR signal near g = 8; this is the first quantitative study of such a signal from any system. Analysis of the temperature dependence of the g = 8 signal yields J = 15 +/- 5 cm-1 for the exchange-coupling constant (H(ex) = JS1.S2). This value is more than 1 order of magnitude smaller than those reported for the oxo-bridged clusters of hemerythrin and Escherichia coli ribonucleotide reductase (H(ex) = JS1.S2, J = 270 and 220 cm-1, respectively), suggesting that the bridging ligand of the hydroxylase cluster is not an unsubstituted oxygen atom. Mossbauer spectra of the hydroxylase in applied fields of up to 8 T reveal a paramagnetic admixture of a low-lying excited state into the ground singlet. Both the spectral shape and intensity are well represented by assuming that the spin expectation values for the cluster sites increase linearly with magnetic field. However, the origin of this effect is not fully explicable in the framework of the standard spin Hamiltonian including zero-field splittings and antisymmetric exchange. EPR studies of the uncomplexed mixed valence (Fe3+.Fe2+) hydroxylase show that it is composed of two slightly different cluster forms in an approximate 4:1 ratio. The zero-field splitting (ZFS) of the ferrous site of the mixed valence hydroxylase is sensitive to complexation with products or inhibitors, while complexation by the component B perturbs the exchange coupling. The binding of the inhibitor dimethyl sulfoxide results in the smallest distribution of ZFS parameters and thus is investigated here in a correlated study using each of the three spectroscopic techniques. The data were analyzed with a spin Hamiltonian that includes exchange coupling (J = 60 cm-1) and mixing of multiplets by zero-field splittings. The analysis shows that the orbital ground state of the ferrous site has predominantly d(xy) symmetry; the z-axis of this orbital points along the z-direction of the cluster g-tensor. Mossbauer and Fe-57-ENDOR spectra indicate that the A-tensor of the ferric site is anisotropic; the Fe-57-ENDOR signals are the first reported for diiron-oxo clusters. Analysis of the Mossbauer spectra of the uncomplexed, reduced (Fe2+.Fe2+) hydroxylase cluster recorded in strong applied fields (up to 6.0 T) unambiguously shows that the two iron sites are inequivalent. Spectra of the oxidized cluster are also best fit by assuming that the irons of the cluster reside in inequivalent environments. Considered in light of the overall two-fold symmetry of hydroxylase revealed by ongoing structural studies, the present findings show that the hydroxylase contains two, probably identical, active-site diiron clusters whose individual iron atoms are structurally distinct. Mossbauer and EPR spectra of the [2Fe-2S]2+,1+ cluster of the MMO reductase component are also reported and analyzed.
  • BG FOX, J SHANKLIN, C SOMERVILLE, E MUNCK
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 6 2486 - 2490 1993年03月 [査読有り][通常論文]
     
    A gene encoding stearoyl-acyl carrier protein DELTA9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mossbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by almost-equal-to 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C-H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.
  • AM ORVILLE, VJ CHEN, A KRIAUCIUNAS, MR HARPEL, BG FOX, E MUNCK, JD LIPSCOMB
    BIOCHEMISTRY 31 19 4602 - 4612 1992年05月 [査読有り][通常論文]
     
    Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Munck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe2+ when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS.ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R. A., Wang, S., Eidsness, M. K., Kriauciunas, A., Frolik, C. A. & Chen, V. J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, Mossbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe2+ ligands in any of these complexes. Spectral quantitations show that the double Cys --> Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe2+ is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS.NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe2+. The EPR spectra of both the IPNS.NO and IPNS.ACV.NO complexes are broadened for samples prepared in O-17-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe2+ coordination of the IPNS.ACV.NO complex accommodates at least three exogenous ligands. Photodissociation of NO from this complex results in a species exhibiting nearly the same Mossbauer spectrum as the IPNS.ACV complex, suggesting that NO addition does not result in a major change in other aspects of the Fe2+ coordination.
  • MP HENDRICH, BG FOX, KK ANDERSSON, PG DEBRUNNER, JD LIPSCOMB
    JOURNAL OF BIOLOGICAL CHEMISTRY 267 1 261 - 269 1992年01月 [査読有り][通常論文]
     
    Electron nuclear double resonance (ENDOR) spectroscopy is used to probe the coordination of the mixed valence (Fe(II).Fe(III)) diiron cluster of the methane monooxygenase hydroxylase component (MMOH-) isolated from Methylosinus trichosporium OB3b. ENDOR resonances are observed along the principal axis directions g1 = 1.94 and g3 = 1.76 from at least nine different protons and two different nitrogens. The nitrogens are strongly coupled and appear to be directly coordinated to the cluster irons. The ratio of their superhyperfine coupling constants is roughly 4:7, which equals the ratio of the spin expectation values of the Fe(II) and Fe(III) in the ground state and suggests that at least one nitrogen is coordinated to each iron of the mixed valence cluster. Moreover, the superhyperfine and quadrupole coupling constants assigned to the Fe(III) site (A(N) = 13.6 MHz, P(N) = 0.7 MHz) are comparable with those observed for semimethemerythrin sulfide (A(N) = 12.1 MHz, P(N) = 0.7 MHz), for which the nitrogen ligands are histidines. At least three of the coupled protons exchange slowly when MMOH- is incubated in D2O, and H-2 ENDOR resonances are subsequently observed. These observations are also consistent with histidine ligation of the iron cluster. On addition of the inhibitor dimethyl sulfoxide (Me2SO) to MMOH- the EPR spectrum sharpens and shifts dramatically. Only one set of N-14 ENDOR resonances is observed with frequencies equal to those assigned to the Fe(III)-histidine resonances of uncomplexed MMOH- suggesting that the nitrogen coordination to the Fe(II) site is altered or possibly lost in the presence of Me2SO. H-2 ENDOR resonances are observed in the presence of d6-Me2SO indicating that the inhibitor Me2SO binds near or possibly to the diiron cluster. In contrast, no H-2 ENDOR resonances are observed from d4-methanol upon addition to MMOH-. Thus, the changes observed in the EPR spectrum of MMOH- upon addition of methanol may result from binding to a site away from the diiron cluster or from bulk solvent effects on the protein structure.
  • BG FOX, Y LIU, JE DEGE, JD LIPSCOMB
    JOURNAL OF BIOLOGICAL CHEMISTRY 266 1 540 - 550 1991年01月 [査読有り][通常論文]
     
    Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha-beta-gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase between shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (K(D) almost-equal-to 0.4-mu-M) or hydroxylase (two binding sites, K(Da) almost-equal-to 10 nM, K(Db) almost-equal-to 8-mu-M). The observed K(D) values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta-subunits, respectively. The alpha and beta-subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occuurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.
  • MP HENDRICH, E MUNCK, BG FOX, JD LIPSCOMB
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 112 15 5861 - 5865 1990年07月 [査読有り][通常論文]
  • BG FOX, JG BORNEMAN, LP WACKETT, JD LIPSCOMB
    BIOCHEMISTRY 29 27 6419 - 6427 1990年07月 [査読有り][通常論文]
  • BG FOX, WA FROLAND, JD LIPSCOMB
    GAS, OIL, AND COAL BIOTECHNOLOGY 1 197 - 214 1990年 [査読有り][通常論文]
  • BG FOX, WA FROLAND, DR JOLLIE, JD LIPSCOMB
    METHODS IN ENZYMOLOGY 188 191 - 202 1990年 [査読有り][通常論文]
  • BG FOX, WA FROLAND, JE DEGE, JD LIPSCOMB
    JOURNAL OF BIOLOGICAL CHEMISTRY 264 17 10023 - 10033 1989年06月 [査読有り][通常論文]
  • R ABE, MS VACCHIO, B FOX, RJ HODES
    NATURE 335 6193 827 - 830 1988年10月 [査読有り][通常論文]
  • Fox BG, Surerus KK, Münck E, Lipscomb JD
    The Journal of biological chemistry 263 22 10553 - 10556 1988年08月 [査読有り][通常論文]
  • BG FOX, JD LIPSCOMB
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 154 1 165 - 170 1988年07月 [査読有り][通常論文]
  • MT STANKOVICH, BG FOX
    BIOCHIMICA ET BIOPHYSICA ACTA 786 1-2 49 - 56 1984年 [査読有り][通常論文]
  • M HUSAIN, MT STANKOVICH, BG FOX
    BIOCHEMICAL JOURNAL 219 3 1043 - 1047 1984年 [査読有り][通常論文]
  • M STANKOVICH, B FOX
    BIOCHEMISTRY 22 19 4466 - 4472 1983年 [査読有り][通常論文]
  • B FOX, CT WALSH
    JOURNAL OF BIOLOGICAL CHEMISTRY 257 5 2498 - 2503 1982年 [査読有り][通常論文]

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