Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc*

Pichia stipitis NAD+-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD+ to NADP+ and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp207, Ile208, Phe209, and Asn211 in the discrimination between NAD+ and NADP+. Single mutants (D207A, I208R, F209S, and N211R) improved 5∼48-fold in catalytic efficiency (kcat/Km) with NADP+ compared with the wild type but retained substantial activity with NAD+. The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in kcat/Km with NADP+, but they still preferred NAD+ to NADP+. The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in kcat/Km with NADP+ than the wild-type enzyme, reaching values comparable with kcat/Km with NAD+ of the wild-type enzyme. Because most NADP+-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP+.

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