Aldehyde Reductase: The Role of C-Terminal Residues in Defining Substrate and Cofactor Specificities

Abstract

The only major structural difference between aldehyde reductase, a primarily NADPH-dependent aldo–keto reductase, and aldose reductase, a dually coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor binding site (C-terminal region) of aldehyde reductase. On the premise that this segment defines the substrate specificity of the enzyme, a mutant of aldehyde reductase lacking residues 306–313 was constructed. In contrast to wild-type enzyme, the mutant enzyme reduced a narrower range of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coenzyme specificity was observed, however, the mutant enzyme being distinctly NADH preferring(Km, NADH= 35 μM, compared to <5 mM for wild-type andKm, NADPH= 670 μM, compared to 35 μM for wild type). Upon analyzing coordinates of aldehyde and aldose reductase, we found that deletion of residues 306–313 may have created a truncated enzyme that retained the three-dimensional structural features of the enzyme's C-terminal segment. The change in substrate specificity could be explained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initiated by the formation of a strong hydrogen bond between Tyr319 and Val300. A similar bond exists in aldose reductase (Tyr309-Ala299). It appears, therefore, that as far as coenzyme specificity is concerned, deletion of residues 306–313 has converted aldehyde reductase into an aldose reductase-like enzyme.