title: Probing AKR1C30 and AKR1C31 with site-directed mutagenesis: identifying the roles of residues 54 and 56 in the binding of substrates and inhibitors.
abstract: Five rabbit aldo-keto reductases (AKRs) that participate in the reduction of drug ketones and endogenous ketosteroids have recently been cloned and characterized. Among them, AKR1C30 and AKR1C31 show the highest amino acid sequence identity of 91%, but markedly differ in their substrate specificity and inhibitor sensitivity. AKR1C30 reduces two drugs (ketotifen and naloxone) and 17-keto-5β-androstanes, whereas AKR1C31 does not reduce the two drugs, but is active towards loxoprofen and various 3/17/20-ketosteroids. In addition, AKR1C30 is selectively inhibited by carbenoxolone, valproic acid and phenobarbital. Residues A54 and R56 are located adjacent to the catalytic residue Y55 of AKR1C30. To clarify the determinants for the substrate specificity and inhibitor sensitivity of AKR1C30, we performed the mutagenesis of A54 and R56 to the corresponding residues (L and Q) of AKR1C31. The A54L mutation produced an enzyme that had almost the same substrate specificity as AKR1C31 and decreased the sensitivity to the inhibitors except for carbenoxolone. The R56Q mutation decreased the affinity for only carbenoxolone among the substrates and inhibitors. Thus, the difference in the properties between the two enzymes can be attributed to their residue difference at positions 54 and 56.
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.forename: Yuki
authors.initials: Y
authors.lastname: Arai
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.forename: Ossama
authors.initials: O
authors.lastname: El-Kabbani
authors.forename: Akira
authors.initials: A
authors.lastname: Hara
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases