Probing AKR1C30 and AKR1C31 with Site-Directed Mutagenesis: Identifying the Roles of Residues 54 and 56 in the Binding of Substrates and Inhibitors

摘要: Five rabbit aldo-keto reductases (AKRs) that participate in the reduction of drug ketones and endogenous ketosteroids have recently been cloned characterized. Among them, AKR1C30 AKR1C31 show highest amino acid sequence identity 91%, but markedly differ their substrate specificity inhibitor sensitivity. reduces two drugs (ketotifen naloxone) 17-keto-5β-androstanes, whereas does not reduce drugs, is active towards loxoprofen various 3/17/20-ketosteroids. In addition, selectively inhibited by carbenoxolone, valproic phenobarbital. Residues A54 R56 are located adjacent to catalytic residue Y55 AKR1C30. To clarify determinants for sensitivity AKR1C30, we performed mutagenesis corresponding residues (L Q) AKR1C31. The A54L mutation produced an enzyme had almost same as decreased inhibitors except carbenoxolone. R56Q affinity only carbenoxolone among substrates inhibitors. Thus, difference properties between enzymes can be attributed at positions 54 56.