Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities.
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 microM, compared to <5 mM for wild-type and Km, NADPH = 670 microM, compared to 35 microM 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.
Rees-Milton
K J
KJ
Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
Jia
Z
Z
Green
N C
NC
Bhatia
M
M
El-Kabbani
O
O
Flynn
T G
TG
D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D007668:Kidney; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine
D010446:Peptide Fragments; D009243:NAD; D009249:NADP; D000449:Aldehyde Reductase