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

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

doi:10.1074/jbc.m801778200

Luigi Di Costanzo, Jason E Drury + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m801778200

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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.

Highlights

AKR1D1 catalyzes a reaction that is unique in steroid enzymology, and it accomplishes this with ease in comparison with chemical methods
Site-directed mutagenesis of these residues to Y58F and E120A supports their role in catalysis one binding and instead remains in the conformation observed in the structure of the substrate-free AKR1D11⁄7NADPϩ complex (Fig. 5)
The x-ray crystal structure of human ⌬4-3-ketosteroid because neither mutant has any detectable activity in the reduc- 5␤-reductase (AKR1D1) is the first structure of a mammalian tion of testosterone using the standard radiometric assay, steroid carbon– carbon double bond reductase

Summary

EXPERIMENTAL PROCEDURES

Materials—The vectors pET16b and pET28a were purchased from Novagen. E. coli strain C41(DE3) was provided by Dr J. Drops containing 4.0 ␮l of enzyme solution (5.0 mg/ml AKR1D1, 2.0 mM NADPϩ, 2.0 mM steroid, and 10.0 mM Tris (pH 7.4)) and 4.0 ␮l of precipitant buffer (0.1 M HEPES/Tris-HCl (pH 7.0), 10 –20% (w/v) polyethylene glycol 4000, and 10% isopropyl alcohol) were equilibrated against a 1-ml reservoir of precipitant buffer. For crystallization of the AKR1D11⁄7NADPϩ complex, hanging drops containing 4.0 ␮l of enzyme solution (5.0 mg/ml AKR1D1, 2 mM NADPH, 2.0 mM 5␤-cholestan-3-one, and 10.0 mM Tris (pH 7.4)) and 4.0 ␮l of precipitant buffer (0.1 M Tris/ HEPES (pH 7.5), 10 –20% (w/v) polyethylene glycol 4000, and 10% isopropyl alcohol) were equilibrated against a 1-ml reservoir of precipitant buffer. The crystal structures of the AKR1D11⁄7NADPϩ1⁄7cortisone, AKR1D11⁄7NADPϩ1⁄7progesterone, AKR1D11⁄7NADPϩ1⁄7HEPES, and TABLE 1

Glycerol Ramachandran statisticse

RESULTS

DISCUSSION