共查询到20条相似文献,搜索用时 15 毫秒
1.
Mattia Pedotti Elena Rosini Gianluca Molla Tommaso Moschetti Carmelinda Savino Beatrice Vallone Loredano Pollegioni 《The Journal of biological chemistry》2009,284(52):36415-36423
Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide. 相似文献
2.
Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis
Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and kcat/Km) of BliGO were 11.22 mM, 0.08 s−1, and 0.01 mM−1 s−1, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM−1 s−1) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency. 相似文献
3.
Olga Khersonsky Orly Dym Colin J. Jackson Dan S. Tawfik 《Journal of molecular biology》2010,396(4):1025-1042
Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge—their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a > 200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a kcat/KM value of ∼ 2600 s− 1 M− 1 and an ∼ 106-fold rate acceleration (kcat/kuncat) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its “quenching” interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pKa of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site. 相似文献
4.
Patricia Molina-Espeja Eva Garcia-Ruiz David Gonzalez-Perez René Ullrich Martin Hofrichter Miguel Alcalde 《Applied and environmental microbiology》2014,80(11):3496-3507
Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships. 相似文献
5.
Sandra Gerstenbruch Hauke Wulf Nina Mu?mann Timothy O’Connell Karl-Heinz Maurer Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2012,96(5):1243-1252
Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity. 相似文献
6.
Thomas Lendrihas Gregory A. Hunter Gloria C. Ferreira 《The Journal of biological chemistry》2010,285(5):3351-3359
5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3–4 Å of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the KmSCoA and kcat values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased kcat without altering KmSCoA. Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved ∼3-fold, whereas that of S254T diminished ∼3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire α-oxoamine synthase family of PLP-dependent enzymes. 相似文献
7.
Improving enzyme properties: when are closer mutations better? 总被引:7,自引:0,他引:7
Study of mutations that improve enzyme properties reveals that in many, but not all, cases closer mutations are more effective than distant ones. For enantioselectivity, substrate selectivity and new catalytic activity (catalytic promiscuity) closer mutations improved enzymes more effectively than distant ones. However, both close and distant mutations can improve activity, thermal stability and also probably stability toward organic solvents. Typical random mutagenesis methods, such as error-prone PCR, create greater numbers of distant mutations than close mutations because enzymes contain more amino acids distant from the active site than close to the active site. This suggests that instead of mutating the entire enzyme, focusing mutations near the substrate-binding site might dramatically increase the success rate in many directed evolution experiments. 相似文献
8.
Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site 总被引:11,自引:0,他引:11
S R Padgette D B Re C S Gasser D A Eichholtz R B Frazier C M Hironaka E B Levine D M Shah R T Fraley G M Kishore 《The Journal of biological chemistry》1991,266(33):22364-22369
The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS. 相似文献
9.
Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique. 相似文献
10.
Kelly RM Leemhuis H Rozeboom HJ van Oosterwijk N Dijkstra BW Dijkhuizen L 《The Biochemical journal》2008,413(3):517-525
Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common 'intermediate-like' ancestor towards specific transglycosylation activity. 相似文献
11.
Doris Ribitsch Sonja Winkler Karl Gruber Wolfgang Karl Eva Wehrschütz-Sigl Inge Eiteljörg Petra Schratl Peter Remler Regina Stehr Cornelius Bessler Nina Mußmann Kerstin Sauter Karl Heinz Maurer Helmut Schwab 《Applied microbiology and biotechnology》2010,87(5):1743-1752
In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme. 相似文献
12.
The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining. 相似文献
13.
Bosko M. Stojanovski Gloria C. Ferreira 《The Journal of biological chemistry》2015,290(52):30750-30761
5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5′-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis. 相似文献
14.
Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L−1 kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively. 相似文献
15.
Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities. 相似文献
16.
Ken'ichiro Matsumoto Yoshikazu Tanaka Tsuyoshi Watanabe Ren Motohashi Koji Ikeda Kota Tobitani Min Yao Isao Tanaka Seiichi Taguchi 《Applied and environmental microbiology》2013,79(19):6134-6139
NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity. 相似文献
17.
Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris 总被引:3,自引:3,他引:0
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Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pKa values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO−CH2NH2+CH2PO32−, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (Ki = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (Ki′ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate]. 相似文献
18.
Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance. 相似文献
19.
A catabolite repression-insensitive mutant of Candida boidinii A5, strain ADU-15, was investigated as to alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production. The mutant strain ADU-15 showed higher cell productivity and higher alcohol oxidase activity when grown on mixed substrates (glucose-methanol), especially with a high concentration of glucose in the medium. Thus, even under substrate (glucose-methanol)-limited chemostat conditions, where the glucose concentration was low, partial derepression of alcohol oxidase by glucose in mutant strain ADU-15 was detected. The chemostat culture conditions with the glucose-methanol medium were optimized for alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production, respectively. By means of chemostat culturing on mixed substrates, we improved the alcohol oxidase productivity 5.0-fold and the productivity of cells exhibiting the maximum catalytic activity for formaldehyde production 3.8-fold, in comparison with the parent strain chemostat cultured with methanol as the single substrate. 相似文献
20.
Gülgez G?k?e Y?ld?z Robert B. Gennis Fevzi Daldal Mehmet ?ztürk 《Journal of bacteriology》2014,196(10):1825-1832
The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb3-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the KC channel) present in the cbb3-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the KC channel of the cbb3-type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the KC channel. 相似文献