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.Key words aldo-keto reductase; carbenoxolone; drug ketone reductase; hydroxysteroid dehydrogenase; sitedirected mutagenesis Carbonyl reduction into the alcohol metabolites is an important Phase-I metabolism of carbonyl-containing drugs. 1,2)To detoxify and excrete the chemically divergent carbonyl compounds, nicotinamide adenine dinucleotide phosphate reduced form (NADPH)-dependent reductases with broad substrate specificity for carbonyl compounds exist in multiple forms in the body. The enzymes with known primary structures are grouped into two distinct protein families: the shortchain dehydrogenase/reductase 1,3) and aldo-keto reductase (AKR) 2-4) superfamilies. Major human enzymes responsible for the reduction of drug ketones in the AKR superfamily are four cytosolic hydroxysteroid dehydrogenase (HSD) isoforms, 2,3) which belong to the AKR1C subfamily and named AKR1C1, AKR1C2, AKR1C3 and AKR1C4.In laboratory animal rats or mice, five NADPH-dependent AKRs similar to the human HSD isoforms were identified and suggested to reduce xenobiotic carbonyl compounds. 3,[5][6][7][8][9][10] In rabbits, such AKRs are six, which are AKR1C5 acting as 17β/20α-HSD, 11,12) AKR1C29 (that is identical to 3-hydroxyhexobarbital dehydrogenase and exhibits 3α/3β/17β/20α-HSD activity), 13) AKR1C30 (that is identical to naloxone reductase type 1 and acts as 17β-HSD), AKR1C31 (that acts as 3α/17β/20α-HSD), AKR1C32 (that is identical to loxoprofen reductase and acts as 3α/20α-HSD) and AKR1C33 (that is identical to naloxone reductase type 2 and mainly acts as 3α-HSD).14) The rabbit AKRs share more than 73% amino acid sequence identity, which is 94% between AKR1C30 and AKR1C31. Despite of the high sequence identity, AKR1C30 and AKR1C31 differ in their specificity for dr...