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The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site.
Biochem J. 2009 Jun 12; 421(1):43-9.BJ

Abstract

Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in alpha-dicarbonyls differs from that utilized in the conversion of xylose.

Authors+Show Affiliations

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

Pub Type(s)

Journal Article

Language

eng

PubMed ID

19368528

Citation

Pival, Simone L., et al. "The Catalytic Mechanism of NADH-dependent Reduction of 9,10-phenanthrenequinone By Candida Tenuis Xylose Reductase Reveals Plasticity in an Aldo-keto Reductase Active Site." The Biochemical Journal, vol. 421, no. 1, 2009, pp. 43-9.
Pival SL, Klimacek M, Nidetzky B. The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site. Biochem J. 2009;421(1):43-9.
Pival, S. L., Klimacek, M., & Nidetzky, B. (2009). The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site. The Biochemical Journal, 421(1), 43-9. https://doi.org/10.1042/BJ20090128
Pival SL, Klimacek M, Nidetzky B. The Catalytic Mechanism of NADH-dependent Reduction of 9,10-phenanthrenequinone By Candida Tenuis Xylose Reductase Reveals Plasticity in an Aldo-keto Reductase Active Site. Biochem J. 2009 Jun 12;421(1):43-9. PubMed PMID: 19368528.
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TY - JOUR T1 - The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site. AU - Pival,Simone L, AU - Klimacek,Mario, AU - Nidetzky,Bernd, Y1 - 2009/06/12/ PY - 2009/4/17/entrez PY - 2009/4/17/pubmed PY - 2009/7/16/medline SP - 43 EP - 9 JF - The Biochemical journal JO - Biochem. J. VL - 421 IS - 1 N2 - Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in alpha-dicarbonyls differs from that utilized in the conversion of xylose. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/19368528/The_catalytic_mechanism_of_NADH_dependent_reduction_of_910_phenanthrenequinone_by_Candida_tenuis_xylose_reductase_reveals_plasticity_in_an_aldo_keto_reductase_active_site_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20090128 DB - PRIME DP - Unbound Medicine ER -