The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

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  • Author(s): Petschacher B;Petschacher B; Leitgeb S; Kavanagh KL; Wilson DK; Nidetzky B
  • Source:
    The Biochemical journal [Biochem J] 2005 Jan 01; Vol. 385 (Pt 1), pp. 75-83.
  • Publication Type:
    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:
    English
  • Additional Information
    • Source:
      Publisher: Published by Portland Press on behalf of the Biochemical Society Country of Publication: England NLM ID: 2984726R Publication Model: Print Cited Medium: Internet ISSN: 1470-8728 (Electronic) Linking ISSN: 02646021 NLM ISO Abbreviation: Biochem J Subsets: MEDLINE
    • Publication Information:
      Original Publication: London, UK : Published by Portland Press on behalf of the Biochemical Society
    • Subject Terms:
      Aldehyde Reductase/*chemistry ; Aldehyde Reductase/*metabolism ; Candida/*enzymology ; Mutagenesis, Site-Directed/*genetics ; NAD/*metabolism ; NADP/*metabolism; Adenosine/metabolism ; Aldehyde Reductase/genetics ; Aldehyde Reductase/isolation & purification ; Candida/genetics ; Catalysis ; Crystallography, X-Ray ; Kinetics ; Models, Molecular ; Mutation/genetics ; Protein Conformation ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Ribose/metabolism ; Substrate Specificity ; Thermodynamics ; Xylose/metabolism
    • 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.
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    • Grant Information:
      R01 GM066135 United States GM NIGMS NIH HHS; GM66135 United States GM NIGMS NIH HHS
    • Molecular Sequence:
      PDB 1SM9
    • Accession Number:
      0 (Recombinant Proteins)
      0U46U6E8UK (NAD)
      53-59-8 (NADP)
      681HV46001 (Ribose)
      A1TA934AKO (Xylose)
      EC 1.1.1.21 (Aldehyde Reductase)
      K72T3FS567 (Adenosine)
    • Publication Date:
      Date Created: 20040824 Date Completed: 20050628 Latest Revision: 20181227
    • Publication Date:
      20221213
    • Accession Number:
      PMC1134675
    • Accession Number:
      10.1042/BJ20040363
    • Accession Number:
      15320875