共查询到15条相似文献,搜索用时 15 毫秒
1.
Carmen Gherasim Markus Ruetz Zhu Li Stephanie Hudolin Ruma Banerjee 《The Journal of biological chemistry》2015,290(18):11393-11402
Human CblC catalyzes the elimination of the upper axial ligand in cobalamin or B12 derivatives entering the cell from circulation. This processing step is critical for assimilation of dietary cobalamin into the active cofactor forms that support the B12-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. Using a modified nitroreductase scaffold tailored to bind cobalamin and glutathione, CblC exhibits versatility in the mechanism by which it removes cyano versus alkyl ligands in cobalamin. In this study, we have characterized the effects of two pathogenic missense mutations at the same residue, R161G and R161Q, which are associated with early and late onset of the CblC disorder, respectively. We find that the R161Q and R161G CblC mutants display lower protein stability and decreased dealkylation but not decyanation activity, suggesting that cyanocobalamin might be therapeutically useful for patients carrying mutations at Arg-161. The mutant proteins also exhibit impaired glutathione binding. In the presence of physiologically relevant glutathione concentrations, stabilization of the cob(II)alamin derivative is observed, which occurs at the expense of increased oxidation of glutathione. Futile redox cycling, which is suppressed in wild-type human CblC, explains the reported increase in oxidative stress levels associated with the CblC disorder. 相似文献
2.
Koen Van Laer Aleksandra M. Dziewulska Marcus Fislage Khadija Wahni Abderahim Hbeddou Jean-Francois Collet Wim Versées Luis M. Mateos Veronica Tamu Dufe Joris Messens 《The Journal of biological chemistry》2013,288(11):7942-7955
NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 106 and 5.6 × 106
m−1 min−1, respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pKa lower than 6.5 for nucleophilic attack, whereas the pKa of the C-terminal cysteine is ∼10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum. 相似文献
3.
Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. 相似文献
4.
Silke Litzinger Stefanie Fischer Patrick Polzer Kay Diederichs Wolfram Welte Christoph Mayer 《The Journal of biological chemistry》2010,285(46):35675-35684
Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain β-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-Å resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His234 of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp232 for proper function. Replacement of both His234 and Asp232 with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4′-methylumbelliferyl N-acetyl-β-d-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and β-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within β-N-acetylglucosaminidases but otherwise absent in β-glycosidases of family 3, which instead carry a “classical” glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His234 of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 β-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of β-lactamase. 相似文献
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6.
Ryuichi Takase Bunzo Mikami Shigeyuki Kawai Kousaku Murata Wataru Hashimoto 《The Journal of biological chemistry》2014,289(48):33198-33214
The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-d-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R′, with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R′ in strain A1, structural determination of A1-R′ by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R′. A1-R′ was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R′. Crystal structures of A1-R′ and A1-R′·NAD+ complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R′ and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2′ region) was observed. Distinct from A1-R, A1-R′ included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R′ mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family. 相似文献
7.
Jeffrey M. Boyd Daniel D. Clark Melissa A. Kofoed Scott A. Ensign 《The Journal of biological chemistry》2010,285(33):25232-25242
The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys82) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys87 was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys82. These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis. 相似文献
8.
Abbas Abou-Hamdan Pierre Ceccaldi Hugo Lebrette Oscar Gutiérrez-Sanz Pierre Richaud Laurent Cournac Bruno Guigliarelli Antonio L. De Lacey Christophe Léger Anne Volbeda Bénédicte Burlat Sébastien Dementin 《The Journal of biological chemistry》2015,290(13):8550-8558
The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production. 相似文献
9.
Bin Deng Sudharsan Parthasarathy WenFang Wang Brian R. Gibney Kevin P. Battaile Scott Lovell David R. Benson Hao Zhu 《The Journal of biological chemistry》2010,285(39):30181-30191
NADH cytochrome b5 oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b5 (b5), CHORD-SGT1 (CS), and cytochrome b5 reductase (b5R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b5 and b5R domains (Ncb5or-b5 and Ncb5or-b5R, respectively) and compared them with human microsomal b5 (Cyb5A) and b5R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b5 reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His89 and His112, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b5 family shown to have such a heme environment. Like other b5 family members, Ncb5or-b5 has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b5 differs from Cyb5A with respect to location of the second heme ligand (His112) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b5R to Ncb5or-b5 is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b5 and b5R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b5R domains suggest that the CS domain facilitates docking of the b5 and b5R domains. Trp114 is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b5R domain to the b5 domain. 相似文献
10.
Ekaterina V. Bobkova Michael J. Weber Zangwei Xu Yan-Ling Zhang Joon Jung Peter Blume-Jensen Alan Northrup Priya Kunapuli Jannik N. Andersen Ilona Kariv 《The Journal of biological chemistry》2010,285(24):18838-18846
The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen® screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, Km values were determined as 5.6 μm for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1× and 10× Km concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1. 相似文献
11.
Yves Bourne Ludovic Renault Pascale Marchot 《The Journal of biological chemistry》2015,290(3):1522-1535
The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. 相似文献
12.
Isamu Z. Hartman Pingsheng Liu John K. Zehmer Katherine Luby-Phelps Youngah Jo Richard G. W. Anderson Russell A. DeBose-Boyd 《The Journal of biological chemistry》2010,285(25):19288-19298
Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets. 相似文献
13.
Li Zhu Linda Millen Juan L. Mendoza Philip J. Thomas 《The Journal of biological chemistry》2010,285(48):37271-37280
Early onset dystonia is commonly associated with the deletion of one of a pair of glutamate residues (ΔE302/303) near the C terminus of torsinA, a member of the AAA+ protein family (ATPases associated with a variety of cellular activities) located in the endoplasmic reticulum lumen. The functional consequences of the disease-causing mutation, ΔE, are not currently understood. By contrast to other AAA+ proteins, torsin proteins contain two conserved cysteine residues in the C-terminal domain, one of which is located in the nucleotide sensor II motif. Depending on redox status, an ATP hydrolysis mutant of torsinA interacts with lamina-associated polypeptide 1 (LAP1) and lumenal domain like LAP1 (LULL1). Substitution of the cysteine in sensor II diminishes the redox-regulated interaction of torsinA with these substrates. Significantly, the dystonia-causing mutation, ΔE, alters the ability of torsinA to mediate the redox-regulated interactions with LAP1 and LULL1. Limited proteolysis experiments reveal redox- and mutation-dependent changes in the local conformation of torsinA as a function of nucleotide binding. These results indicate that the cysteine-containing sensor II plays a critical role in redox sensing and the nucleotide and partner binding functions of torsinA and suggest that loss of this function of torsinA contributes to the development of DYT1 dystonia. 相似文献
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Nagashima K Shumway SD Sathyanarayanan S Chen AH Dolinski B Xu Y Keilhack H Nguyen T Wiznerowicz M Li L Lutterbach BA Chi A Paweletz C Allison T Yan Y Munshi SK Klippel A Kraus M Bobkova EV Deshmukh S Xu Z Mueller U Szewczak AA Pan BS Richon V Pollock R Blume-Jensen P Northrup A Andersen JN 《The Journal of biological chemistry》2011,286(8):6433-6448
Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems. 相似文献