MUTATIONAL ANALYSIS OF THE ROLE OF THE CONSERVED LYSINE-270 IN THE PICHIA-STIPITIS XYLOSE REDUCTASE

Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose t o xylitol and is essential for growth on xylose by yeasts. To understa nd the nature of coenzyme binding to the Pichia stipitis xylose reduct ase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5 -16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained un changed. This resulted in 3.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-f old decrease in K-m for xylose, but only small changes were observed i n K-m values for glyceraldehyde. The wild-type enzyme, but not the Lys 270Met variant, was susceptible to modification by the Lys-specific py ridoxal 5'-phosphate. Results of our chemical modification and site-di rected mutagenesis study indicated that Lys270 is involved in both NAD PH and D-xylose binding in the P. stipitis xylose reductase. (C) 1998 Federation of European Microbiological Societies. Published by Elsevie r Science B.V.