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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.

Original publication

DOI

10.1074/jbc.m202160200

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

07/2002

Volume

277

Pages

25677 - 25684

Addresses

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

Keywords

Oxidoreductases, DNA Primers, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Amino Acid Sequence, Base Sequence, Protein Folding, Sequence Homology, Amino Acid, Structure-Activity Relationship, Kinetics, Catalysis, Models, Molecular, Molecular Sequence Data