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J Biol Chem. 2008 Jun 13;283(24):16830-9. doi: 10.1074/jbc.M801778200. Epub 2008 Apr 11.

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

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1
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.

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.

PMID:
18407998
PMCID:
PMC2423251
DOI:
10.1074/jbc.M801778200
[Indexed for MEDLINE]
Free PMC Article
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