Biochem. J. (2005) 385
(75–83) (Printed in Great Britain)
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography
Barbara PETSCHACHER*, Stefan LEITGEB*†, Kathryn L. KAVANAGH†, David K. WILSON† and Bernd NIDETZKY*1
*Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria, and †Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, U.S.A.
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.
Key words: aldo–keto reductase, coenzyme selectivity, NADH, NADPH, site-directed mutagenesis, xylose fermentation.
Abbreviations used: AKR, aldo–keto reductase; CtXR, candida tenuis xylose reductase; 2,5-DKG, 2,5-diketogluconate reductase; Mut, mutant; R.M.S.D., root mean square deviation; WT, wild-type; XDH, xylitol dehydrogenase.
1To whom correspondence should be addressed (email bernd.nidetzky@tugraz.at).
Co-ordinates of the reported protein structure have been deposited in the Protein Data Bank (PDB) under the accession number 1SM9.
Received 5 March 2004/2 August 2004; accepted 23 August 2004
Published as BJ Immediate Publication 23 August 2004, DOI 10.1042/BJ20040363
The Biochemical Society, London ©2005
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