Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types |
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Authors: | Mario Klimacek Margarete Szekely Richard Grießler Bernd Nidetzky |
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Institution: | Institute of Food Technology, University of Agricultural Sciences (BOKU), Muthgasse 18, A-1190, Vienna, Austria. |
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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 (Tyrn-(X)3-Lysn+4) and aldo/keto reductases (AKRs) (Tyrn-(X)28-Lysn+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
-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. |
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Keywords: | Aldo/keto reductase Single-domain reductase/epimerase/dehydrogenase Catalytic tyrosine |
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