Crystal Structure Determination and Mutagenesis Analysis of the Ene Reductase NCR
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The crystal structure of the "ene" nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β-unsaturated alkenes, the rational design methods site- and loop-directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single-residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop-swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild-type NCR towards the set of substrates tested. The active-site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop-swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild-type NCR ene reductase enzyme.Keywords:
Site-directed mutagenesis
Ene reaction
Based on the experimental data and homologous sites in Protein Data Bank (PDB) a model for metal binding sites in D1/D2 heterodimer has been proposed. On searching for tetranuclear and binuclear Mn binding sites in the PDB, a suitable sequence homology in thermolysin and D1 could be observed. From the homology and site-directed mutagenesis data, a model for binuclear Mn-Ca or Mn-Mn has been built and it is extended to a tetranuclear Mn centre.
Thermolysin
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Abstract Penicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three‐dimensional structure is not known. Based upon the X‐ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three‐dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall β‐jelly roll folding common to family 11 xylanases with two large β‐pleated sheets and a single α‐helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the “thumb” and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico ‐based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three‐dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site‐directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44 PgXynA and Ser129 PgXynA , two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by −1 to +0.5 units by mutating Ser44 PgXynA to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K m and V max whereas a S44A mutant lost both pH‐dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP‐I. The replacement of Gln121 PgXynA , located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity. Proteins 2008. © 2008 Wiley‐Liss, Inc.
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Site-directed mutagenesis
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Ramachandran plot
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Cordyceps militaris
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Carbonic anhydrase II
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Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and / or bioremediation.
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Abstract Considering the risk represented by plague today as a potential biological warfare agent, we propose cytosolic Yersinia pestis dihydrofolate reductase (Yp DHFR) as a new target to the design of selective plague chemotherapy. This enzyme has a low homology with the human enzyme and its crystallographic structure has been recently deposited in the Protein Data Bank (PDB). Comparisons of the docking energies and molecular dynamic behaviors of five known DHFR inhibitors inside a 3D model of Yp DHFR (adapted from the crystallographic structure) and human DHFR (Hss DHFR), revealed new potential interactions and suggested insights into the design of more potent Hss DHFR inhibitors as well as selective inhibitors for Yp DHFR. Key words: Plague Yersinia pestis Yp DHFRHomology ModelingDockingMolecular DynamicsSelective inhibition.
Yersinia pestis
Dihydrofolate reductase
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plague
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Three-dimensional structure model of azoreductase AZR of Rhodobacter sphaeroides was con- structed using homology modeling method. It is a flavodoxin adopting α/β structure. Structure alignment of two different types of flavin-dependent azoreductases revealed that they possessed high similarity. Based on sequence and structure analysis, site-directed mutagenesis of K109H and K109A were performed. The opti- mal pH values are pH 6 and pH 9 for K109H and K109A mutant protein, respectively. The optimal tempera- ture (30℃) is not affected by mutagenesis. Positively charged residues at position 109 is necessary for the binding of methyl red, while K109H is not a conserved mutagenesis for the binding of NADPH. K109 may only be involved in the binding of the 2’-phosphate group of NADPH and have no effect on the binding of NADH.
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Rhodobacter sphaeroides
Flavodoxin
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Rhodobacter
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The three-dimensional (3D) structure of proteins is necessary to understand the properties and functions of proteins. Determining protein structure by laboratory equipment is quite complicated and expensive. An alternative method to predict the 3D structure of proteins in the in silico method. One of the in silico methods is homology modeling. Homology modeling is done using the SWISS-MODEL server. Proteins that will be modeled in the 3D structure are proteins that do not yet have a structure in the RCSB PDB database. Protein sequences were obtained from the UniProt database with code A0A0B6VWS2. The results showed that there were two models selected, namely model-1 with the PDB code template 1q0e and model-2 with the PDB code template 3gtv. The results of sequence alignment and model visualization show that model-1 and model-2 are identical. The evaluation and assessment of model-1 on the Ramachandran Plot have a Favored area of ??97.36%, a MolProbity score of 0.79, and a QMEAN value is 1.13. Model-1 is a good 3D protein structure model.
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