AMINO-ACID SUBSTITUTIONS IN THE YEAST PICHIA-STIPITIS XYLITOL DEHYDROGENASE COENZYME-BINDING DOMAIN AFFECT THE COENZYME SPECIFICITY

Directed mutagenesis has been used to identify a set of amino acids in the Pichia stipitis xylitol dehydrogenase, encoded by the xylitol deh ydrogenase gene XYL2, which is involved in specific NAD binding. Withi n the binding domain, a characteristic beta alpha beta-fold is centere d around a glycine motif GXGXXG also containing conserved aspartate an d lysine/arginine residues. The mutation D207-->G and the double mutat ion D207-->G and D210-->G increased the apparent K-m for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respe ctively, as compared to the unaltered enzyme. The introduction of the potential NADP-recognition sequence (GSRPVC) of the alcohol dehydrogen ase from Thermoanaerobium brockii into the xylitol dehydrogenase allow ed the mutant enzyme to use both NAD and NADP as cofactor with equal a pparent K-m values. Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably e xpressed enzyme variant is an important first step towards the long-te rm goal to reverse the coenzyme specificity from NAD to NADP. The muta genized XYL2 gene could still mediate growth of Saccharomyces cerevisi ae transformants on xylose minimal-medium plates when expressed togeth er with the xylose reductase gene (XYL1).