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. 2002 Sep 15;366(Pt 3):889–899. doi: 10.1042/BJ20020080

Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis.

Peter Mayr 1, Bernd Nidetzky 1
PMCID: PMC1222813  PMID: 12003638

Abstract

Kinetic substituent effects have been used to examine the catalytic reaction profile of xylose reductase from the yeast Candida tenuis, a representative aldo/keto reductase of primary carbohydrate metabolism. Michaelis-Menten parameters (k(cat) and K(m)) for NADH-dependent enzymic aldehyde reductions have been determined using a homologous series of benzaldehyde derivatives in which substituents in meta and para positions were employed to systematically perturb the properties of the reactive carbonyl group. Kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m) for enzymic reactions with meta-substituted benzaldehydes have been obtained by using NADH (2)H-labelled in the pro-R C4-H position, and equilibrium constants for the conversion of these aldehydes into the corresponding alcohols (K(eq)) have been measured in the presence of NAD(H) and enzyme. Aldehyde dissociation constants (K(d)) and the hydride transfer rate constant (k(7)) have been calculated from steady-state rate and KIE data. Quantitative structure-activity relationship analysis was used to factor the observed substituent dependence of k(cat)/K(m) into a major electronic effect and a productive positional effect of the para substituent. k(cat)/K(m) (after correction for substituent position) and K(eq) obeyed log-linear correlations over the substituent parameter, Hammett sigma, giving identical slope values (rho) of +1.4 to +1.7, whereas the same Hammett plot for logK(d) yielded rho=-1.5. This leads to the conclusion that electron-withdrawing substituents facilitate the reaction and increase binding to about the same extent. KIE values for k(cat) (1.8) and k(cat)/K(m) (2.7), and likewise k(7), showed no substituent dependence. Therefore, irrespective of the observed changes in reactivity over the substrate series studied no shift in the character of the rate-limiting transition state of hydride transfer occurred. The signs and magnitudes of rho values suggest this transition state to be product-like in terms of charge development at the reactive carbon. Structure-reactivity correlations reveal active-site homologies among NADPH-specific and dual NADPH/NADH-specific yeast xylose reductases and across two aldo/keto reductase families in spite of the phylogenetic separation of the host organisms producing xylose reductase (family 2B) and aldehyde reductase (family 1A).

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Selected References

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  1. Aristidou A., Penttilä M. Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol. 2000 Apr;11(2):187–198. doi: 10.1016/s0958-1669(00)00085-9. [DOI] [PubMed] [Google Scholar]
  2. Bhatnagar A., Liu S. Q., Srivastava S. K. Structure-activity correlations in human kidney aldehyde reductase-catalyzed reduction of para-substituted benzaldehyde by 3-acetyl pyridine adenine dinucleotide phosphate. Biochim Biophys Acta. 1991 Apr 8;1077(2):180–186. doi: 10.1016/0167-4838(91)90056-6. [DOI] [PubMed] [Google Scholar]
  3. Bohren K. M., Grimshaw C. E., Lai C. J., Harrison D. H., Ringe D., Petsko G. A., Gabbay K. H. Tyrosine-48 is the proton donor and histidine-110 directs substrate stereochemical selectivity in the reduction reaction of human aldose reductase: enzyme kinetics and crystal structure of the Y48H mutant enzyme. Biochemistry. 1994 Mar 1;33(8):2021–2032. doi: 10.1021/bi00174a007. [DOI] [PubMed] [Google Scholar]
  4. Grimshaw C. E. Aldose reductase: model for a new paradigm of enzymic perfection in detoxification catalysts. Biochemistry. 1992 Oct 27;31(42):10139–10145. doi: 10.1021/bi00157a001. [DOI] [PubMed] [Google Scholar]
  5. Grimshaw C. E., Bohren K. M., Lai C. J., Gabbay K. H. Human aldose reductase: pK of tyrosine 48 reveals the preferred ionization state for catalysis and inhibition. Biochemistry. 1995 Nov 7;34(44):14374–14384. doi: 10.1021/bi00044a014. [DOI] [PubMed] [Google Scholar]
  6. Jez J. M., Penning T. M. The aldo-keto reductase (AKR) superfamily: an update. Chem Biol Interact. 2001 Jan 30;130-132(1-3):499–525. doi: 10.1016/s0009-2797(00)00295-7. [DOI] [PubMed] [Google Scholar]
  7. Klimacek M., Szekely M., Griessler R., Nidetzky B. Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types. FEBS Lett. 2001 Jul 6;500(3):149–152. doi: 10.1016/s0014-5793(01)02609-6. [DOI] [PubMed] [Google Scholar]
  8. Klinman J. P. Isotope effects and structure-reactivity correlations in the yeast alcohol dehydrogenase reaction. A study of the enzyme-catalyzed oxidation of aromatic alcohols. Biochemistry. 1976 May 4;15(9):2018–2026. doi: 10.1021/bi00654a032. [DOI] [PubMed] [Google Scholar]
  9. Klinman J. P. The mechanism of enzyme-catalyzed reduced nicotinamide adenine dinucleotide-dependent reductions. Substituent and isotope effects in the yeast alcohol dehydrogenase reaction. J Biol Chem. 1972 Dec 25;247(24):7977–7987. [PubMed] [Google Scholar]
  10. Kresge A. J., Silverman D. N. Application of Marcus rate theory to proton transfer in enzyme-catalyzed reactions. Methods Enzymol. 1999;308:276–297. doi: 10.1016/s0076-6879(99)08014-3. [DOI] [PubMed] [Google Scholar]
  11. Lee Y. S., Hodoscek M., Brooks B. R., Kador P. F. Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics. Biophys Chem. 1998 Mar 9;70(3):203–216. doi: 10.1016/s0301-4622(97)00115-4. [DOI] [PubMed] [Google Scholar]
  12. Ma H., Ratnam K., Penning T. M. Mutation of nicotinamide pocket residues in rat liver 3 alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding. Biochemistry. 2000 Jan 11;39(1):102–109. doi: 10.1021/bi991659o. [DOI] [PubMed] [Google Scholar]
  13. Mayr P., Brüggler K., Kulbe K. D., Nidetzky B. D-Xylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with different coenzyme specificities. J Chromatogr B Biomed Sci Appl. 2000 Jan 14;737(1-2):195–202. doi: 10.1016/s0378-4347(99)00380-1. [DOI] [PubMed] [Google Scholar]
  14. Neuhauser W., Haltrich D., Kulbe K. D., Nidetzky B. NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme. Biochem J. 1997 Sep 15;326(Pt 3):683–692. doi: 10.1042/bj3260683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neuhauser W., Haltrich D., Kulbe K. D., Nidetzky B. Noncovalent enzyme-substrate interactions in the catalytic mechanism of yeast aldose reductase. Biochemistry. 1998 Jan 27;37(4):1116–1123. doi: 10.1021/bi9717800. [DOI] [PubMed] [Google Scholar]
  16. Nidetzky B., Klimacek M., Mayr P. Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis. Biochemistry. 2001 Aug 28;40(34):10371–10381. doi: 10.1021/bi010148a. [DOI] [PubMed] [Google Scholar]
  17. Nidetzky B., Mayr P., Hadwiger P., Stütz A. E. Binding energy and specificity in the catalytic mechanism of yeast aldose reductases. Biochem J. 1999 Nov 15;344(Pt 1):101–107. [PMC free article] [PubMed] [Google Scholar]
  18. Nidetzky B., Mayr P., Neuhauser W., Puchberger M. Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis. Chem Biol Interact. 2001 Jan 30;130-132(1-3):583–595. doi: 10.1016/s0009-2797(00)00285-4. [DOI] [PubMed] [Google Scholar]
  19. Northrop D. B. Deuterium and tritium kinetic isotope effects on initial rates. Methods Enzymol. 1982;87:607–625. doi: 10.1016/s0076-6879(82)87032-8. [DOI] [PubMed] [Google Scholar]
  20. Penning T. M. Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase. J Steroid Biochem Mol Biol. 1999 Apr-Jun;69(1-6):211–225. doi: 10.1016/s0960-0760(99)00038-2. [DOI] [PubMed] [Google Scholar]
  21. Schlegel B. P., Jez J. M., Penning T. M. Mutagenesis of 3 alpha-hydroxysteroid dehydrogenase reveals a "push-pull" mechanism for proton transfer in aldo-keto reductases. Biochemistry. 1998 Mar 10;37(10):3538–3548. doi: 10.1021/bi9723055. [DOI] [PubMed] [Google Scholar]
  22. Tarle I., Borhani D. W., Wilson D. K., Quiocho F. A., Petrash J. M. Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110. J Biol Chem. 1993 Dec 5;268(34):25687–25693. [PubMed] [Google Scholar]

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