title: The active site architecture of a short-chain dehydrogenase defined by site-directed mutagenesis and structure modeling.
abstract: A high-resolution crystal structure is not currently available for Drosophila alcohol dehydrogenase. A detailed three-dimensional model for this enzyme, based on the structure of 3 alpha,20 beta-hydroxysteroid dehydrogenase, has been generated by extensive computer modeling studies. Aspects of the model concerned with coenzyme binding have been tested by site-directed mutagenesis of residues Gly-14 to Ala, Gly-19 to Ala, Asp-38 to Ala, and Pro-214 to Ser. All enzymes have been characterized in terms of kinetic constants, relative stabilities to guanidinium chloride, and heat inactivation. The contribution of NAD binding to the stabilization of each of the enzymes was also measured. The results obtained with enzymes mutated at positions 14, 38, and 214 are in accordance with published data on Drosophila alcohol dehydrogenase and suggest interactions of these residues with the cofactor NAD. The introduction of a methyl group at residue Gly-19 abolished the ability of the enzyme to utilize NADP instead of NAD. This reflects a proximity of residue Gly-19 to the ribose ring of the bound cofactor. This result, coupled to the three-dimensional model built for Drosophila alcohol dehydrogenase, suggests a binding mechanism for the cofactor NAD different from that found for 3 alpha,20 beta-dehydroxysteroid dehydrogenase and similar to that found in the crystal structure of rat liver dihydropteridine reductase. The model of Drosophila alcohol dehydrogenase also enables many previous observations from chemical modification, sequence comparisons, site-directed mutagenesis, and limited proteolysis experiments to be placed into a structural context. An active site architecture is proposed involving a loop closure mechanism similar to that of lactate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Edinburgh, Scotland.
authors.forename: L
authors.initials: L
authors.lastname: Ribas de Pouplana
authors.forename: L A
authors.initials: LA
authors.lastname: Fothergill-Gilmore
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D004330:Drosophila / Q000201:enzymology; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D006146:Guanidines; D009243:NAD; D000426:Alcohol Dehydrogenase; D019791:Guanidine