共查询到20条相似文献,搜索用时 15 毫秒
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Anne L Carenbauer James D Garrity Gopal Periyannan Robert B Yates Michael W Crowder 《BMC biochemistry》2002,3(1):4-13
Background
The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. 相似文献3.
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Nadya G Gurskaya Alexander P Savitsky Yurii G Yanushevich Sergey A Lukyanov Konstantin A Lukyanov 《BMC biochemistry》2001,2(1):6-7
Background
Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters. 相似文献5.
Rational control of enantioselectivity of lipase by site-directed mutagenesis based on the mechanism
Ema T Fujii T Ozaki M Korenaga T Sakai T 《Chemical communications (Cambridge, England)》2005,(37):4650-4651
The enantioselectivity of a Burkholderia cepacia lipase toward secondary alcohols could be both increased and decreased rationally by introducing only a single mutation on the basis of the mechanism proposed previously. 相似文献
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Resonance energy transfer between tryptophanyl residues and the apolar fluorescent dye 1-anilino-8-naphthalene sulfonate (ANS) occurs when the fluorophore is bound to native folded sperm whale apomyoglobin. The individual transfer contribution of the two tryptophanyl residues (W7 and W14, both located on the A-helix of the protein) was resolved by measuring the tryptophan-ANS transfer efficiency for the ANS-apomyoglobin complexes formed by wild-type protein and protein mutants containing one or no tryptophanyl residues, i.e. W7F, W14F and W7YW14F. The transfer efficiency of W14 residue was found to be higher than that of W7, thus indicating that W14 acts as the main energy donor in the ANS-apomyoglobin complex. This suggests that the plane containing the anilinonaphthalene ring of the extrinsic fluorophore has a spatial orientation similar to that of W14 and, hence, to the heme group in the holoprotein. 相似文献
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Seemann M Zhai G de Kraker JW Paschall CM Christianson DW Cane DE 《Journal of the American Chemical Society》2002,124(26):7681-7689
Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in k(cat) and k(cat)/K(m). By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg(2+)-binding aspartate-rich domain, (80)DDLFD, resulted in reduction in the k(cat)/K(m) for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k(cat)/K(m) which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product beta-caryophyllene (9) (9%). Finally, the F77Y mutant had a k(cat)/K(m) which was reduced by 20-fold compared to that of the wild-type synthase. 相似文献
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BACKGROUND: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. RESULTS: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the omega end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. CONCLUSIONS: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms. 相似文献
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L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD+ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for
the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition
can be overcome by weaking the binding of NAD+.
The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based
overlap extension mutagenesis technique in the hope of weakening NAD+-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate
inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold. 相似文献
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Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4). 相似文献
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Koo LS Immoos CE Cohen MS Farmer PJ Ortiz de Montellano PR 《Journal of the American Chemical Society》2002,124(20):5684-5691
CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids. 相似文献
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Ijima Y Matoishi K Terao Y Doi N Yanagawa H Ohta H 《Chemical communications (Cambridge, England)》2005,(7):877-879
The introduction of two mutations (G74C/C188S) based on the estimated reaction mechanism resulted in the inversion of enantioselectivity of arylmalonate decarboxylase, which catalyses the asymmetric decarboxylation of arylmethylmalonate to give optically active arylpropionate. 相似文献
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Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis 总被引:2,自引:0,他引:2
Busca P Paradisi F Moynihan E Maguire AR Engel PC 《Organic & biomolecular chemistry》2004,2(18):2684-2691
The substrate scope of three mutants of phenylalanine dehydrogenase as biocatalysts for the transformation of a series of 2-oxo acids, structurally related to phenylpyruvic acid, to the analogous alpha-amino acids, non-natural analogues of phenylalanine, has been investigated. The mutant enzymes are more tolerant than the wild type enzyme of the non-natural substrates, especially those with substituents at the 4-position on the phenyl ring. Excellent enantiocontrol resulted in all cases. 相似文献
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Soujanya D. Yelamanchi Sumaithangi Thattai Arun Kumar Archita Mishra Thottethodi Subrahmanya Keshava Prasad Avadhesha Surolia 《Molecules (Basel, Switzerland)》2022,27(5)
Mycobacterium tuberculosis has been infecting millions of people worldwide over the years, causing tuberculosis. Drugs targeting distinct cellular mechanisms including synthesis of the cell wall, lipids, proteins, and nucleic acids in Mtb are currently being used for the treatment of TB. Although extensive research is being carried out at the molecular level in the infected host and pathogen, the identification of suitable drug targets and drugs remains under explored. Pranlukast, an allosteric inhibitor of MtArgJ (Mtb ornithine acetyltransferase) has previously been shown to inhibit the survival and virulence of Mtb. The main objective of this study was to identify the altered metabolic pathways and biological processes associated with the differentially expressed metabolites by PRK in Mtb. Here in this study, metabolomics was carried out using an LC-MS/MS-based approach. Collectively, 50 metabolites were identified to be differentially expressed with a significant p-value through a global metabolomic approach using a high-resolution mass spectrometer. Metabolites downstream of argJ were downregulated in the arginine biosynthetic pathway following pranlukast treatment. Predicted human protein interactors of pranlukast-treated Mtb metabolome were identified in association with autophagy, inflammation, DNA repair, and other immune-related processes. Further metabolites including N-acetylglutamate, argininosuccinate, L-arginine, succinate, ergothioneine, and L-phenylalanine were validated by multiple reaction monitoring, a targeted mass spectrometry-based metabolomic approach. This study facilitates the understanding of pranlukast-mediated metabolic changes in Mtb and holds the potential to identify novel therapeutic approaches using metabolic pathways in Mtb. 相似文献
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Towards the proteome of Mycobacterium tuberculosis 总被引:14,自引:0,他引:14
Rosenkrands I King A Weldingh K Moniatte M Moertz E Andersen P 《Electrophoresis》2000,21(17):3740-3756
Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis. Sequencing of the genome of M. tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting. Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two-dimensional gel electrophoresis using immobilized pH gradient technology. By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel. To date, 288 proteins have been identified in M. tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at http://www.ssi.dk/publichealth/tbimmun). The information obtained from the M. tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence. 相似文献
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Petzold CJ Stanton LH Leary JA 《Journal of the American Society for Mass Spectrometry》2005,16(7):1109-1116
Structural aspects of lipoarabinomannans (LAM) from Mycobacterium tuberculosis and Mycobacterium smegmatis were investigated by using mild acid hydrolysis in combination with Fourier-transform ion cyclotron resonance (FT-ICR), and quadrupole ion trap mass spectrometry. Exact mass measurements with less than 2.5 ppm mass error confirmed the presence of a series of arabinose oligomers (Ara(n); n = 2-7) as the major components observed following mild acid hydrolysis of both M. tuberculosis and M. smegmatis LAM. However, the mass spectrum of the resulting LAM extract also revealed a highly-abundant distribution of ions that exact mass measurements identified as mannose-linked arabinose species, Ara(n)Man(m) + Na+ (n = 1-6; m = 1-3). The observed mannose caps were linked to arabinose species as mono-, di-, and trimannose units, and the ratio of the mono-, di-, and trimannose caps was determined to be 1.00:9.00:1.15, respectively, different from previous reports. Analysis of the linkage of lithiated arabinose trimer standards was accomplished with MS3 experiments and the information generated was used to identify linkages of arabinose trimers generated by mild acid hydrolysis of M. tuberculosis and M. smegmatis LAM. The MS3 spectra confirmed the linkage of arabinose trimers from M. smegmatis and M. tuberculosis LAM as predominantly alpha(1 --> 5), alpha(1 --> 5). 相似文献
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Nakamura T Motoyama T Hirokawa T Hirono S Yamaguchi I 《Chemical & pharmaceutical bulletin》2003,51(11):1293-1298
Homology modeling was used to construct a model of the three-dimensional structure of pentachlorophenol 4-monooxygenase (PcpB). A PSI-BLAST homology search was initially performed to identify the 3D structure of proteins homologous with PcpB. The feasibility of modeled structures of PcpB was evaluated by Verify3D, which calculated structural compatibility scores based on 3D-1D profiles. The predicted structure of PcpB had an acceptable 3D-1D self-compatibility score, beyond the incorrect fold score threshold. A PcpB-pentachlorophenol (PCP) complex was then constructed utilizing the modeled PcpB structure. After energy minimization of the complex, and successive minimizations of the system that consisted of the complex and the water layer surrounding the complex, the molecular dynamics of the system were simulated. The active-site residues of PcpB were identified on the basis of the modeled structure, and PcpB mutants were then designed to change the active site residues, expressed, and purified by affinity chromatography. The mutant activity was compared with that of the wild-type to investigate the validity of the modeled structure. The experimental results suggested that Phe85, Tyr216, and Arg235 were relevant to enzyme activity, and that Tyr397 and Phe87 were important for stabilization of the structure of PcpB. 相似文献
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J Wen S W Nelson R B Honzatko H J Fromm J W Petrich 《Photochemistry and photobiology》2001,74(5):679-685
The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan. 相似文献