ALDEHYDE REDUCTASE - THE ROLE OF C-TERMINAL RESIDUES IN DEFINING SUBSTRATE AND COFACTOR SPECIFICITIES

The only major structural difference between aldehyde reductase, a pri marily NADPH-dependent aldo-keto reductase, and aldose reductase, a du ally coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor bi nding site (C-terminal region) of aldehyde reductase. On the premise t hat this segment defines the substrate specificity of the enzyme, a mu tant of aldehyde reductase lacking residues 306-313 was constructed. I n contrast to wild-type enzyme, the mutant enzyme reduced a narrower r ange of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coen zyme specificity was observed, however, the mutant enzyme being distin ctly NADH preferring (K-m,K- NADH = 35 mu M, compared to <5 mM for wil d-type and K-m,K- NADPH = 670 mu M, compared to 35 mu M for Wild type) . Upon analyzing coordinates of aldehyde and aldose reductase, we foun d that deletion of residues 306-313 may have created a truncated enzym e that retained the three-dimensional structural features of the enzym e's C-terminal segment. The change in substrate specificity could be e xplained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initia ted by the formation of a strong hydrogen bond between Tyr319 and Val3 00. A similar bond exists in aldose reductase (Tyr309-Ala299). It appe ars, therefore, that as far as coenzyme specificity is concerned, dele tion of residues 306-313 has converted aldehyde reductase into an aldo se reductase-like enzyme. (C) 1998 Academic Press.