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

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of sing le-domain reductases/epimerases/dehydrogenases (Tyr(n)-(X)(3)-Lys(n+4)) and aldo/keto reductases (AKRs) (Tyr(n)-(X)(28)-Lys(n-29)). Tyr(51), Lys(55) a nd Lys(80) of XR from Candida tenuis were replaced by site-directed mutagen esis, The purified Tyr(51) --> Phe and Lys(80) --> Ala mutants showed turno ver numbers and catalytic efficiencies for NADH-dependent reduction of D-xy lose between 2500- and 5000-fold below wild-type levels, suggesting a catal ytic role of both residues, Replacing Lys(55) by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymati c catalysis to carbonyl/alcohol interconversion, The contribution of Tyr(51 ) to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction, (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rig hts reserved.