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 |
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Authors: | Petschacher Barbara Nidetzky Bernd |
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Institution: | Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. |
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Abstract: | 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. |
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