Enzyme Catalysis and Regulation
Crystal Structure of Human Liver Δ4-3-Ketosteroid 5β-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis*

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AKR1D1 (steroid 5β-reductase) reduces all Δ4-3-ketosteroids to form 5β-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 α,β-unsaturated ketone by 5β-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the β-face of a Δ4-3-ketosteroid yields a cis-A/B-ring configuration with an ∼90° bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Δ4-3-ketosteroid 5β-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP+ at 1.79- and 1.35-Å resolution (HEPES bound in the active site), NADP+ and cortisone at 1.90-Å resolution, NADP+ and progesterone at 2.03-Å resolution, and NADP+ and testosterone at 1.62-Å 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 Tyr58 and Glu120. 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.

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This paper is dedicated to Dr. Paul Talalay, The John Jacob-Abel Distinguished Service Professor, Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, in honor of his 85th birthday. Paul has been a pioneer in steroid hormone enzymology throughout his career.

The atomic coordinates and structure factors (code 3BUR, 3CMF, 3COT, 3BUV, and 3BV7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported, in whole or in part, by National Institutes of Health Research Grant R01-GM56838 (to D. W. C.) and Grants R01-DK47015 and P30-ES013508 (to T. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

This article was selected as a Paper of the Week.