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

Simone L. Pival , Mario Klimacek , Bernd Nidetzky (1)

1 TU Graz - Graz University of Technology [Graz]

Despite their widely varying physiological functions in carbonyl metabolism, Candida tenuis xylose reductase (AKR2B5) and many related enzymes of the aldo-keto reductase protein superfamily utilise 9,10 phenanthrenequinone (PQ) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80, His113) of AKR2B5 in the conversion of the reactive α-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by Ala) 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 103 – 104-fold decrease in the rate constant for hydride transfer from NADH to PQ whose value in the wildtype enzyme was determined as ~8 x 102 s-1. The presented data 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 α- 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 catalysis to carbonyl group reduction in α- dicarbonyls differs from that utilised in the conversion of xylose.

Mots clés: Life Sciences

Simone L. Pival, Mario Klimacek, Bernd Nidetzky. 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. Biochemical Journal, 2009, 421 (1), pp.43-49. ⟨10.1042/BJ20090128⟩. ⟨hal-00479157⟩