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

Journal Article · Tue Jul 15 00:00:00 EDT 2008 · J. Biol. Chem.

DOI:https://doi.org/10.1074/jbc.M801778200· OSTI ID:1006656

Di Costanzo, Luigi; Drury, Jason E; Penning, Trevor M; Christianson, David W ^[1]

---

1. UPENN

AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 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 {alpha}{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 {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} 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}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} 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{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 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.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: USDOE
• OSTI ID: 1006656
• Journal Information: J. Biol. Chem., Vol. 283, Issue (24) ; 06, 2008; ISSN 0021-9258
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
BILE ACIDS
CARBONYLS
CATALYSIS
CLEARANCE
COMPLEXES
CONFIGURATION
CORTISONE
CRYSTAL STRUCTURE
DOUBLE BONDS
HUMAN POPULATIONS
HYDRIDES
HYDROGEN
INHIBITION
KETONES
LIVER
MUTANTS
MUTATIONS
NICOTINAMIDE
ORIENTATION
PROGESTERONE
REDUCTION
RESIDUES
RESOLUTION
RINGS
STEROID HORMONES
STEROIDS
SUBSTRATES
SYNTHESIS
TESTOSTERONE