title: Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+.
abstract: Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD) inactivates circulating steroid hormones and is involved in polycyclic aromatic hydrocarbon (PAH) carcinogenesis. It is the only HSD of known structure in the aldo-keto reductase (AKR) superfamily and may provide a paradigm for other mammalian HSDs in this family. The structure of the 3 alpha-HSD.NADP+ binary complex has been determined at 2.7 A resolution and refined to a crystallographic R-factor of 23.4% with good geometry. The model is similar to other binary complexes in the AKR superfamily in that NADP+ binds at the C-terminal end of an alpha/beta barrel. However, it is unique in that NADP+ is bound in two alternate conformations, probably because of the lack of a salt-linked "safety belt" over the pyrophosphate bridge. The structure supports a previously proposed catalytic mechanism for carbonyl reduction in which Tyr 55 is the general acid, and its effective pKa is lowered by the adjacent Lys 84. We present evidence that the structurally distinct short-chain dehydrogenase/reductase (SDR) superfamily may have convergently evolved a similar catalytic mechanism. Insight into substrate binding is offered by a crystal packing contact in which a neighboring molecule inserts a tryptophan residue (Trp 227) into an apolar cleft in 3 alpha-HSD. This cleft is proximal to the bound NADP+ cofactor and contains a surface of apolar residues (Leu 54, Trp 86, Leu 122, Phe 128, Phe 129, Leu 137, Phe 139), making it a likely candidate for the substrate-binding site. Thus, in forming this crystal contact, Trp 227 may mimic a portion of a bound steroid. In addition, we propose that a water molecule in the active site indicates the position of the hydroxyl oxygen in a 3 alpha-hydroxysteroid substrate. Knowledge of the position of this water molecule, combined with the stereochemistry of hydride transfer, suggests that the alpha face of a bound steroid will be oriented toward the side of the apolar cleft containing Trp 86.
authors.affiliation: Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia 19104-6059, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Bennett
authors.forename: B P
authors.initials: BP
authors.lastname: Schlegel
authors.forename: J M
authors.initials: JM
authors.lastname: Jez
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
authors.forename: M
authors.initials: M
authors.lastname: Lewis
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D008099:Liver / Q000201:enzymology; D008297:Male; D008958:Models, Molecular; D015394:Molecular Structure; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013256:Steroids / Q000378:metabolism
chemical_list: D013256:Steroids; D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases