title: Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase.
abstract: Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, 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 unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5'-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and D-xylose binding in the P. stipitis xylose reductase.
authors.affiliation: Department of Environmental Biology, University of Guelph, Ont., Canada.
authors.forename: M
authors.initials: M
authors.lastname: Kostrzynska
authors.forename: C R
authors.initials: CR
authors.lastname: Sopher
authors.forename: H
authors.initials: H
authors.lastname: Lee
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000032:analysis; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D004271:DNA, Fungal; D011732:Pyridoxal Phosphate; D000449:Aldehyde Reductase; D008239:Lysine