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, Bernd Nidetzky

Research output: Contribution to journalArticle

96 Scopus citations

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 °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.

Original languageEnglish (US)
Pages (from-to)75-83
Number of pages9
JournalBiochemical Journal
Volume385
Issue number1
DOIs
StatePublished - Jan 1 2005

Keywords

  • Aldo-keto reductase
  • Coenzyme selectivity
  • NADH
  • NADPH
  • Site-directed mutagenesis
  • Xylose fermentation

ASJC Scopus subject areas

  • Biochemistry

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