Crystal structure of human liver Δ4-3-ketosteroid 5β-reductase (AKR1D1) and implications for substrate binding and catalysis

Abstract

AKR1D1 (steroid 5β-reductase) reduces all Δ4-3- ketosteroids to form 5β-dihydrosteroids, a first step in the clearance of steroid hormonesandanessentialstepinthesynthesisofallbileacids.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 amammaliansteroid 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.