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 reductases (AKRs) that determine their substrate specificities it would be desirable 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 structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing 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 complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major 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 through hydrogen bonding to the tyrosine hydroxyl group.