The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto redu ctases (AKRs) that determine their substrate specificities it would be desi rable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP (H) and substrate or product have not been achieved. Previous crystal struc tures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general ac id-base catalytic residue in the enzyme. This view is supported by the gene ration of an enzymatically inactive site-directed mutant (tyrosine-48 to ph enylalanine) in human aldose reductase [AKR1B1]. However, crystallization o f this mutant was unsuccessful. We have attempted to generate a trapped cof actor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the g eneration of the first high resolution binary AKR-Y50F:NADP(H) crystal stru cture, but we were unable to generate any ternary complexes. The binary com plex was refined to 2.2A and shows a clear lack of density due to the missi ng hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures; The mutant binds cofactor (both oxidized and reduced) more tightly but shows a comple te lack of binding of the aldehyde reductase inhibitor barbitone as determi ned by fluorescence titrations. Attempts at substrate addition to the activ e site, either by cocrystallization or by soaking, were all unsuccessful us ing pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, meth ylglyoxal, and other substrates. The lack of ternary complex formation, com bined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the act ive site tyrosine is essential for substrate binding in addition to its maj or role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate throug h hydrogen bonding to the tyrosine hydroxyl group. (C) 2001 Elsevier Scienc e Ireland Ltd. All rights reserved.