Exploring the active site of yeast xylose reductase by site‐directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

FEBS Letters

Szekely, Margarete¹  Grießler, Richard¹  Klimacek, Mario¹  Nidetzky, Bernd¹ 

[1] Institute of Food Technology, University of Agricultural Sciences (BOKU), Muthgasse 18, A-1190 Vienna, Austria

关键词: Aldo/keto reductase;    Single-domain reductase/epimerase/dehydrogenase;    Catalytic tyrosine;    XR;    xylose reductase;    AKR;    aldo/keto reductase;    RED;    single-domain reductase/epimerase/dehydrogenase;    hAR;    human aldose reductase;    PCR;    polymerase chain reaction;    3α-HSD;    3α-hydroxysteroid dehydrogenase;    CtXR;    xylose reductase from Candida tenuis;   

DOI  :  10.1016/S0014-5793(01)02609-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyr ⁿ -(X)₃-Lys ⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyr ⁿ -(X)₂₈-Lys ⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of D-xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.

【 授权许可】

Unknown