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 dehydrogenase gene XYL2, which is involved in specific NAD binding. Within the binding domain, a characteristic beta alpha beta-fold is centered around a glycine motif GXGXXG also containing conserved aspartate and lysine/arginine residues. The mutation D207-->G and the double mutation D207-->G and D210-->G increased the apparent Km for NAD ninefold and decreased the xylitol dehydrogenase activity to 47% and 35%, respectively, as compared to the unaltered enzyme. The introduction of the potential NADP-recognition sequence (GSRPVC) of the alcohol dehydrogenase from Thermoanaerobium brockii into the xylitol dehydrogenase allowed the mutant enzyme to use both NAD and NADP as cofactor with equal apparent Km values. Although this mutant enzyme displayed an unaltered NADP acceptance, the reduction of the NAD specificity in the stably expressed enzyme variant is an important first step towards the long-term goal to reverse the coenzyme specificity from NAD to NADP. The mutagenized XYL2 gene could still mediate growth of Saccharomyces cerevisiae transformants on xylose minimal-medium plates when expressed together with the xylose reductase gene (XYL1).
Metzger
M H
MH
Institut für Mikrobiologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Hollenberg
C P
CP
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D001665:Binding Sites; D050542:D-Xylulose Reductase; D005980:Glutathione Reductase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000378:metabolism*
D009243:NAD; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D000426:Alcohol Dehydrogenase; D050542:D-Xylulose Reductase; D005980:Glutathione Reductase