title: Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids.
abstract: Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.
authors.affiliation: Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) 2705 boul. Laurier, Ste-Foy, Qc., Canada G1V 4G2.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Couture
authors.forename: Pierre
authors.initials: P
authors.lastname: Legrand
authors.forename: Line
authors.initials: L
authors.lastname: Cantin
authors.forename: Van
authors.initials: V
authors.lastname: Luu-The
authors.forename: Fernand
authors.initials: F
authors.lastname: Labrie
authors.forename: Rock
authors.initials: R
authors.lastname: Breton
mesh_terms: D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D003460:Crystallization; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011374:Progesterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012399:Rotation; D013237:Stereoisomerism; D013256:Steroids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D008024:Ligands; D013256:Steroids; D011374:Progesterone; D006639:Histidine; D009249:NADP; D001120:Arginine; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D007930:Leucine