Engineering Candida tenuis Xylose reductase for improved utilization of NADH: antagonistic effects of multiple side chain replacements and performance of site-directed mutants under simulated in vivo conditions.
Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2'-phosphate binding pocket were tested for NADPH versus NADH selectivity (R(sel)) in the presence of physiological reactant concentrations. The experimental R(sel) values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274-->Arg and Arg-280-->His substitutions, which individually improved wild-type R(sel) 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.
Petschacher
Barbara
B
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Nidetzky
Bernd
B
D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
D003067:Coenzymes; D009243:NAD; D014994:Xylose; D000449:Aldehyde Reductase