title: Critical residues for structure and catalysis in short-chain dehydrogenases/reductases.
abstract: Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Filling
authors.forename: Kurt D
authors.initials: KD
authors.lastname: Berndt
authors.forename: Jordi
authors.initials: J
authors.lastname: Benach
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
authors.forename: Tim
authors.initials: T
authors.lastname: Prozorovski
authors.forename: Erik
authors.initials: E
authors.lastname: Nordling
authors.forename: Rudolf
authors.initials: R
authors.lastname: Ladenstein
authors.forename: Hans
authors.initials: H
authors.lastname: Jörnvall
authors.forename: Udo
authors.initials: U
authors.lastname: Oppermann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D010088:Oxidoreductases