1r38

Crystal structure of H114A mutant of Candida tenuis xylose reductase

spinqualitylabelsall models 1r38, resolution 2.20Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 1r38 is a 4 chain structure with sequence from Yamadazyma tenuis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.2Å Ligands: NAP Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function XYL1_CANTE Reduces D-xylose into xylitol. Has a preference for NADPH, but can also utilize NADH as cosubstrate. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex. Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.,Kratzer R, Kavanagh KL, Wilson DK, Nidetzky B Biochemistry. 2004 May 4;43(17):4944-54. PMID:15109252 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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