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The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.
Biochem J. 2005 Jan 01; 385(Pt 1):75-83.BJ

Abstract

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 degrees 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 A resolution.

Authors+Show Affiliations

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15320875

Citation

Petschacher, Barbara, et al. "The Coenzyme Specificity of Candida Tenuis Xylose Reductase (AKR2B5) Explored By Site-directed Mutagenesis and X-ray Crystallography." The Biochemical Journal, vol. 385, no. Pt 1, 2005, pp. 75-83.
Petschacher B, Leitgeb S, Kavanagh KL, et al. The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. Biochem J. 2005;385(Pt 1):75-83.
Petschacher, B., Leitgeb, S., Kavanagh, K. L., Wilson, D. K., & Nidetzky, B. (2005). The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. The Biochemical Journal, 385(Pt 1), 75-83.
Petschacher B, et al. The Coenzyme Specificity of Candida Tenuis Xylose Reductase (AKR2B5) Explored By Site-directed Mutagenesis and X-ray Crystallography. Biochem J. 2005 Jan 1;385(Pt 1):75-83. PubMed PMID: 15320875.
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TY - JOUR T1 - The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. AU - Petschacher,Barbara, AU - Leitgeb,Stefan, AU - Kavanagh,Kathryn L, AU - Wilson,David K, AU - Nidetzky,Bernd, PY - 2004/8/24/pubmed PY - 2005/6/29/medline PY - 2004/8/24/entrez SP - 75 EP - 83 JF - The Biochemical journal JO - Biochem. J. VL - 385 IS - Pt 1 N2 - 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 degrees 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 A resolution. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/15320875/The_coenzyme_specificity_of_Candida_tenuis_xylose_reductase__AKR2B5__explored_by_site_directed_mutagenesis_and_X_ray_crystallography_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20040363 DB - PRIME DP - Unbound Medicine ER -