Mh. Metzger et Cp. Hollenberg, AMINO-ACID SUBSTITUTIONS IN THE YEAST PICHIA-STIPITIS XYLITOL DEHYDROGENASE COENZYME-BINDING DOMAIN AFFECT THE COENZYME SPECIFICITY, European journal of biochemistry, 228(1), 1995, pp. 50-54
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).