CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.
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Research Article|
December 14 2004
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography
Barbara PETSCHACHER;
Barbara PETSCHACHER
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
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Stefan LEITGEB;
Stefan LEITGEB
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
†Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
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Kathryn L. KAVANAGH;
Kathryn L. KAVANAGH
†Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
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David K. WILSON;
David K. WILSON
†Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
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Bernd NIDETZKY
Bernd NIDETZKY
1
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
1To whom correspondence should be addressed (email bernd.nidetzky@tugraz.at).
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Publisher: Portland Press Ltd
Received:
March 05 2004
Revision Received:
August 02 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 75–83.
Article history
Received:
March 05 2004
Revision Received:
August 02 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Citation
Barbara PETSCHACHER, Stefan LEITGEB, Kathryn L. KAVANAGH, David K. WILSON, Bernd NIDETZKY; The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. Biochem J 1 January 2005; 385 (1): 75–83. doi: https://doi.org/10.1042/BJ20040363
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