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1.
Previously reported rates of reaction between six mutant strains of the enzyme horseradish peroxidase (HRP) and a test substrate, 2-methoxyphenol, were found to correlate with characteristic binding distances computed using molecular simulation. The correlation (R(2) = 0.86) bears out a working hypothesis that, based on a quantitative structure-activity relationship (QSAR) we had previously developed for HRP, reductions in binding distances between the HRP enzyme and any selected substrate mediate increased enzyme reactivity towards that substrate. The results validate the use of QSAR as a quantitative means for formulating enzyme mutations designed to achieve enhanced HRP reactivity towards compounds of specific interest. 相似文献
2.
Vinh H. Nguyen Noreen Singh Ana Medina Isabel Usn Marie E. Fraser 《Protein science : a publication of the Protein Society》2019,28(10):1840-1849
ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes. 相似文献
3.
Margarita Kanteev Noam Adir 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(3):237-242
The cyanobacterium Synechocystis sp. PCC 6803 imports Mn2+ ions via MntCAB, an ABC transport system that is expressed at submicromolar Mn2+ concentrations. The structures of the wild type (WT) and a site‐directed mutant of the MntC solute‐binding protein have been determined at 2.7 and 3.5 Å resolution, respectively. The WT structure is significantly improved over the previously determined structure (PDB entry 1xvl ), showing improved Mn2+ binding site parameters, disulfide bonds in all three monomers and ions bound to the protein surface, revealing the role of Zn2+ ions in the crystallization liquor. The structure of MntC reveals that the active site is surrounded by neutral‐to‐positive electrostatic potential and is dominated by a network of polar interactions centred around Arg116. The mutation of this residue to alanine was shown to destabilize loops in the entrance to the metal‐ion binding site and suggests a possible role in MntC function. 相似文献
4.
Sireesha Ratakonda Arvind Anand Kanak Dikshit Benjamin C. Stark Andrew J. Howard 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(3):215-222
Site‐directed mutants of the gene encoding wild‐type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild‐type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild‐type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild‐type VHb as determined by CO‐difference spectra. Circular‐dichroism spectra also showed the two mutants to be essentially the same as wild‐type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7–1.9 Å, which are similar to that of the original wild‐type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild‐type VHb as well as in the wild‐type VHb structure reported here. In the Tyr29Ala mutant, the five‐membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wild‐type structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen‐binding pocket. 相似文献
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6.
Yonca Yuzugullu Chi H. Trinh Mark A. Smith Arwen R. Pearson Simon E. V. Phillips Didem Sutay Kocabas Ufuk Bakir Zumrut B. Ogel Michael J. McPherson 《Acta Crystallographica. Section D, Structural Biology》2013,69(3):398-408
Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon‐optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis‐hydroxychlorin γ‐spirolactone, which is rotated 180° with respect to small‐subunit catalases. The haem‐binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d‐type haem to a b‐type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide. 相似文献
7.
Artem Y. Lyubimov Lin Chen Nicole S. Sampson Alice Vrielink 《Acta Crystallographica. Section D, Structural Biology》2009,65(11):1222-1231
Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3β‐hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH...π interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800‐fold drop in the overall kcat. Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild‐type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild‐type Asn485 forms the NH...π interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered. 相似文献
8.
Grundman O Khozin-Goldberg I Raveh D Cohen Z Vyazmensky M Boussiba S Shapira M 《Biotechnology and bioengineering》2012,109(9):2340-2348
Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type. 相似文献
9.
Dawei Fu Huoqing Huang Kun Meng Yaru Wang Huiying Luo Peilong Yang Tiezheng Yuan Bin Yao 《Biotechnology and bioengineering》2009,103(5):857-864
A new phytase (APPA) with optimum pH 2.5—substantially lower than that of most of microbial phytases (pH 4.5–6.0)—was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site‐directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side‐chain structure near the active site—contributes to the optimal pH for APPA activity. Compared with wild‐type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild‐type APPA for use in animal feed. Biotechnol. Bioeng. 2009;103: 857–864. © 2009 Wiley Periodicals, Inc. 相似文献
10.
The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity. 相似文献
11.
Zhang X Li T Zhang J Li D Guo Y Qin G Zhu KY Ma E Zhang J 《Archives of insect biochemistry and physiology》2012,80(2):77-91
Glutathione S-transferases (GSTs) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8-anilino-1-naphthalenesulfonic acid (ANS) florescence results showed that the two tyrosine residues were involved in regulation of active-site conformation. Finally, homology modeling provided evidence that the two tyrosines in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability. 相似文献
12.
Kithalakshmi Vignesvaran Zazali Alias 《Archives of insect biochemistry and physiology》2016,92(3):210-221
Drosophila melanogaster glutathione S‐transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N‐terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in Km (twofold increase), Vmax (fivefold reduction), and Kcat/Km values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1‐chloro‐2,4‐dinitrobenzene as no significant change in values of Kmax was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (Kcat/Km)GSH and (Kcat/Km)CDNB of eight‐ and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2‐dichloro‐4‐nitrobenzene, 2,4‐hexadienal, 2,4‐heptadienal, p‐nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation. 相似文献
13.
A. Ghelfi S.A. Gaziola M.C. Cia S.M. Chabregas M.C. Falco P.R. Kuser‐Falcão R.A. Azevedo 《The Annals of applied biology》2011,159(2):267-280
Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1‐chloro‐2,4‐dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron‐sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different Km values not only for CDNB, but also for GSH. The Km values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The Vmax values were 297.6, 224.5 and 171.8 µmol min?1 mg?1 protein for GSH, and 372.3, 170.6 and 160.4 µmol min?1 mg?1 protein for CDNB. 相似文献
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15.
Tomschy A Tessier M Wyss M Brugger R Broger C Schnoebelen L van Loon AP Pasamontes L 《Protein science : a publication of the Protein Society》2000,9(7):1304-1311
Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction. 相似文献
16.
Cesari F Rennekampff V Vintersten K Vuong LG Seibler J Bode J Wiebel FF Nordheim A 《Genesis (New York, N.Y. : 2000)》2004,38(2):87-92
Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome. 相似文献
17.
丁酰胆碱酯酶(butyrylcholinesterase, BChE, EC 3.1.1.8),能与有机磷毒剂或杀虫剂结合,并能水解许多酯类、肽类及酰胺类化合物,对这些化合物的中毒具有防治作用.近年来通过计算机模拟技术及定点突变技术对其结构研究取得了重要进展,对人BChE外周阴离子部位的结构有了新的认识, 并通过氨基酸替换使BChE获得了水解有机磷酸酐的新功能. 相似文献
18.
V. A. Mitkevich A. A. Schulga A. A. Trofimov P. V. Dorovatovskii D. A. Goncharuk E. N. Tkach A. A. Makarov K. M. Polyakov 《Acta Crystallographica. Section D, Structural Biology》2013,69(6):991-996
Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three‐dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi‐1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7‐fold and its cytotoxic properties are enhanced compared with the wild‐type enzyme. 相似文献
19.
Chih‐Jung Kuo Chun‐Hsiang Huang Yingying Zheng Tzu‐Ping Ko Zhen Zhu Xinxin Feng Ke Wang Eric Oldfield Andrew H.‐J. Wang Po‐Huang Liang Rey‐Ting Guo Yanhe Ma 《Proteins》2015,83(1):37-45
Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C40 long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc. 相似文献
20.
Lidia Rodríguez‐Fernndez F. Javier Lpez‐Jaramillo Adelbert Bacher Markus Fischer Sevil Weinkauf 《Acta Crystallographica. Section F, Structural Biology Communications》2008,64(7):625-628
Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150 Å icosahedral capsid consisting of 60 subunits and crystallizes in space group P6322 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4 Å resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit‐cell parameters a = b = 313.02, c = 365.77 Å, α = β = 90.0, γ = 120°, and diffracts to 1.6 Å resolution. 相似文献