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National Institute of Environmental Health Sciences

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Publication Detail

Title: Crystal structure of human liver Delta4-3-ketosteroid 5beta-reductase (AKR1D1) and implications for substrate binding and catalysis.

Authors: Di Costanzo, Luigi; Drury, Jason E; Penning, Trevor M; Christianson, David W

Published In J Biol Chem, (2008 Jun 13)

Abstract: AKR1D1 (steroid 5beta-reductase) reduces all Delta(4)-3-ketosteroids to form 5beta-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an alpha,beta-unsaturated ketone by 5beta-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the beta-face of a Delta(4)-3-ketosteroid yields a cis-A/B-ring configuration with an approximately 90 degrees bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Delta(4)-3-ketosteroid 5beta-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP(+) at 1.79- and 1.35-A resolution (HEPES bound in the active site), NADP(+) and cortisone at 1.90-A resolution, NADP(+) and progesterone at 2.03-A resolution, and NADP(+) and testosterone at 1.62-A resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP(+). This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr(58) and Glu(120). The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

PubMed ID: 18407998 Exiting the NIEHS site

MeSH Terms: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/chemistry; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/metabolism*; Binding Sites; Carbon/chemistry; Catalysis; Cortisone/chemistry; Crystallography, X-Ray/methods; Humans; Kinetics; Liver/enzymology*; Models, Chemical; Molecular Conformation; Mutation; Progesterone/chemistry; Protein Binding; Steroids/chemistry

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