title: Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types.
abstract: 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)). Tyr(51), Lys(55) and Lys(80) of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr(51)--> Phe and Lys(80)-->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 Lys(55) 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 Tyr(51) to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.
authors.affiliation: Institute of Food Technology, University of Agricultural Sciences (BOKU), Muthgasse 18, A-1190, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Klimacek
authors.forename: M
authors.initials: M
authors.lastname: Szekely
authors.forename: R
authors.initials: R
authors.lastname: Griessler
authors.forename: B
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry* / Q000235:genetics*; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002175:Candida / Q000201:enzymology*; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014994:Xylose / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase