Human cytosolic hydroxysteroid dehydrogenases of the aldo-ketoreductase superfamily catalyze reduction of conjugated steroids: implications for phase I and phase II steroid hormone metabolism

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.

SCHEME 1.

Examples of steroid transformation reactions catalyzed by AKR1C isoforms: inactivation of Dht and activation of Tib.

SCHEME 2.

Structures of the steroid conjugates studied.

FIGURE 1.

LC/MS analysis of the reduction of DhtG catalyzed by human AKR1C isoforms. A, the ion chromatogram (m/z 450-500) of a mixture of authentic standards of DhtG and 3α-Diol-17-G; B-F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1-4 (C-F). Samples were prepared as described under “Experimental Procedures.”

FIGURE 2.

LC/MS analysis of the reduction of DhtS catalyzed by human AKR1C isoforms. A, the ion chromatogram (m/z 350-400) of a mixture of authentic standards of DhtS and 3β-Diol-17-S; B-F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1-4 (C-F). Samples were prepared as described under “Experimental Procedures.”

FIGURE 3.

LC/MS analysis of the reduction of TibS catalyzed by human AKR1C isoforms. A, the ion chromatogram (m/z 200-400) of a mixture of authentic standards of TibS, 3α-OH-TibS, and 3β-OH-TibS; B-F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1-4 (C-F). Samples were prepared as described under “Experimental Procedures.”

FIGURE 4.

The steroid binding site of AKR1C enzymes can accommodate conjugated steroids. A binding model of DhtG in AKR1C2 is shown. The details of the model building are given under “Experimental Procedures.” The (α/β)₈-barrel structure of AKR1C2 is shown in ribbon representation with α-helices in purple and β-strands in yellow. NADP⁺, shown in stick representation in orange, binds in a highly conserved position. DhtG, shown in sphere representation with carbon in green and oxygen in red, binds in the steroid binding site that is formed by residues from several loops (in blue). The 3-keto group of the steroid is in close proximity with the nicotinamide ring of the cofactor for 3-ketosteroid reduction. The conjugate group of the steroid is accommodated in the wide opening at the entry point of the steroid binding pocket. The closest neighboring residues (in light blue) to the glucuronide group are shown in stick and dot representations. The figure was prepared using PyMOL.

SCHEME 3.

Conjugation pathway to 3α-Diol-17-G predominates in liver.

SCHEME 4.

Reduction pathway to 3α-Diol-17-G predominates in prostate. RL-HSD, RoDH Like 3α-HSD.

SCHEME 5.

Role of human AKR1C isoforms in the metabolism of Tib.