Crystal Structure of Human Liver delta {4}-3-Ketosteroid 5 beta-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

Journal Article · Tue Jan 01 04:00:00 EST 2008 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M801778200· OSTI ID:959582

Di Costanzo, L; Drury, J; Penning, T; Christianson, D

AKR1D1 (steroid 5{beta}-reductase) reduces all 4-3-ketosteroids to form 5{beta}-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 a,{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a 4-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}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 4-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP+ at 1.79- and 1.35- Angstroms resolution (HEPES bound in the active site), NADP+ and cortisone at 1.90- Angstroms resolution, NADP+ and progesterone at 2.03- Angstroms resolution, and NADP+ and testosterone at 1.62- Angstroms 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.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 959582
• Report Number(s): BNL--82568-2009-JA
• Journal Information: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 24 Vol. 283; ISSN JBCHA3; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

08 HYDROGEN
36 MATERIALS SCIENCE
BILE ACIDS
CARBONYLS
CATALYSIS
CLEARANCE
CONFIGURATION
CORTISONE
CRYSTAL STRUCTURE
DOUBLE BONDS
HYDRIDES
HYDROGEN
KETONES
LIVER
NICOTINAMIDE
PROGESTERONE
RESIDUES
RESOLUTION
STEROID HORMONES
SUBSTRATES
SYNTHESIS
TESTOSTERONE
national synchrotron light source