title: Glutamate-115 renders specificity of human 11beta-hydroxysteroid dehydrogenase type 2 for the cofactor NAD+.
abstract: The renal 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 catalyzes the NAD(+)-dependent oxidation of the C11-alcohol on cortisol and corticosterone to yield inactive 11-ketosteroids. The lack of purified active enzyme complicates structure-function analyses of 11beta-HSD2. Here, we constructed a 3D-structural model of 11beta-HSD2, based on known 3D-structures of other short-chain dehydrogenases/reductases (SDR), and functionally analyzed 11beta-HSD2 mutants predicted to be involved in cofactor binding. Our 3D-model explains the preference for NAD(+) over NADP(+) by the coulombic repulsion between the adenosine ribose 2'-phosphate on NADP(+) and the carboxylate on Glu(115) and to steric hindrance with the side chain on Glu(115). Indeed, replacement of Glu(115) with serine or threonine, lacking repulsive charge and unfavorable steric interactions, showed only 3-fold preference for NAD(+), compared to 40-fold for wild-type 11beta-HSD2. Mutation of both Asp(91) and Glu(115) to serine raised NADP(+)-dependent activity to that with NAD(+), but caused reduced enzymatic activity. The 3D-model predicted that this is due to a loss of stabilizing interactions of Asp(91) with Cys(90), Glu(115), Asn(117) and Gly(120). Thus, predictions using the 3D-model combined with analysis of mutants allowed the identification of residues critical for NAD(+)-dependent activity of 11beta-HSD2.
authors.affiliation: Division of Nephrology and Hypertension, Department of Clinical Research, University of Berne, Freiburgstrasse 15, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Arnold
authors.forename: Steven
authors.initials: S
authors.lastname: Tam
authors.forename: Lisa
authors.initials: L
authors.lastname: Yan
authors.forename: Michael E
authors.initials: ME
authors.lastname: Baker
authors.forename: Felix J
authors.initials: FJ
authors.lastname: Frey
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D003198:Computer Simulation; D005971:Glutamates / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D005971:Glutamates; D009243:NAD; D001224:Aspartic Acid; D010649:Phenylalanine; D009249:NADP; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2