Abstract
Aldo-ketoreductase 1C (AKR1C) enzymes catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids. They are Phase I metabolizing enzymes for natural and synthetic steroid hormones. They convert 5alpha-dihydrotestosterone (Dht, potent androgen) to 3alpha/beta-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3alpha/beta-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17beta-glucuronide (DhtG), Dht-17beta-sulfate (DhtS), and Tib-17beta-sulfate (TibS). Product identities were characterized by liquid chromatography-mass spectrometry, and kinetic parameters of the reactions were determined. The product profile of the reduction of each steroid conjugate by the individual AKR1C isoform was similar to that of the corresponding free steroid except for the reduction of DhtG catalyzed by AKR1C2, where a complete inversion in stereochemical preference to 3beta-reduction (with DhtG) from 3alpha-reduction (with Dht and DhtS) was observed. The catalytic efficiency of 3-keto reduction was modestly affected by the presence of a 17-sulfate group but severely impaired by the presence of a 17-glucuronide group for AKR1C1-3 isoforms. AKR1C4, however, showed superior catalytic efficiencies versus the other isoforms, and those were unaffected by steroid conjugation. Our findings provide evidence for alternative pathways of steroid metabolism where the phase I reaction (reduction) occurs after the phase II reaction (conjugation). Specifically, it is indicated that Dht is metabolized to its metabolite 3alpha-androstanediol-17-glucuronide via the previously unrecognized "conjugation pathway" involving the sequential reactions of UGT2B17 and AKR1C4 in liver but via the conventional "reduction pathway" involving the sequential reactions of AKR1C2 and UGT2B15/17 in prostate.
Highlights
Aldo-ketoreductase (AKR)2 1C enzymes are cytosolic hydroxysteroid dehydrogenases (HSDs) that catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids [1]
The reduction of Tib catalyzed by AKR1C1–3 was rather stereospecific in comparison to the reduction of Dht by Aldo-ketoreductase 1C (AKR1C) isoforms
The activities of human AKR1C enzymes on steroid hormone substrates have been thoroughly studied to show that in vivo these enzymes can regulate the metabolism of androgens, estrogens, and progestins via their 3, 17, and 20-ketosteroid reductase activities [2, 5]
Summary
The 17-position on the stereochemistry and the catalytic efficiency of 3-ketosteroid reduction. Product Profiles of Reduction of DhtG Catalyzed by AKR1C1– 4—Enzymatic activities were observed by LC/MS analysis when AKR1C enzymes were incubated with DhtG. The product profiles for the reduction of DhtG and Dht catalyzed by AKR1C enzymes were compared (Table 1). AKR1C4 has high catalytic efficiency for the turnover of the free and conjugated Dht. In the Tib series, comparable kinetics were observed for the reduction of the free and conjugated steroids (Table 3). When the reduction of DhtG is compared with Dht, severely impaired catalytic efficiencies were observed with AKR1C1 and AKR1C2, and a complete inversion of stereochemistry was seen with AKR1C2 These experimental observations suggest that the accommodation of the 17-glucuronide group of a steroid substrate by these enzymes significantly affects the binding of the 3-keto group. Because it is known that the binding sites of AKR1C enzymes undergo significant conformational change when the binary complex is converted to the ternary complex [18, 19], docking of the steroid conjugates into AKR1C4 was not further pursued
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