Catalytic characteristics of AKR1C1-AKR1C4 and AKR1C9 on oxymesterone.

• AKR1Cs display 5α-reductase activities towards oxymesterone, and the catalytic activities of AKR1C9 and AKR1C4 are higher than those of AKR1C1, AKR1C2 and AKR1C3. • 5α-reductase activities of AKR1Cs towards oxymesterone were higher than towards 17α-methyltestosterone, likely due to the presence of the C4‑hydroxyl group. • In contrast to 4-hydroxytestosterone, AKR1Cs has no 3α/β-HSD activity towards oxymesterone, likely due to the presence of the C17α-methyl group. • Oxymesterone was interconverted with 17α-methyltestosterone by both dehydroxylation and hydroxylation reactions catalyzed by AKR1Cs. The function of aldo-keto reductases (AKRs) is highly variable and is controlled by various groups within the steroid structure. The representative AKRs are AKR1C1-AKR1C4 from humans and AKR1C9 from rats (AKR1Cs). In this study, we focused on how the C4‑hydroxyl in the A-ring and C17α-methyl in the d-ring of oxymesterone (OXY) affect the catalytic function of AKR1Cs. We found that OXY bound to all AKR1Cs with greater affinity for AKR1C9 and AKR1C2, according to both autodocking predictions and binding experiments using microscale thermophoresis. Conversion and enzyme kinetic studies showed that AKR1C4 and AKR1C9 catalyzed approximately 90 % of OXY and that their Kcat and Kcat/Km values were higher than other AKR1Cs. Moreover, both purified AKR1C enzymes and AKR1C-transfected cells reduced OXY to 17α-methyl-4ξ,17β-dihydroxy-androstane-3-one with 5α-reductase activity, that was promoted by C4‑hydroxyl. Nevertheless, AKR1Cs lost the 3α/3β-hydroxysteroid dehydrogenase activity for OXY due to C17α-methyl and did not form 17α-methyl-3ξ, 4ξ, 17β-trihydroxy-androstane. [Display omitted] [ABSTRACT FROM AUTHOR]