Human Cytosolic Hydroxysteroid Dehydrogenases of the Aldo-ketoreductase Superfamily Catalyze Reduction of Conjugated Steroids

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 5␣-dihydrotestosterone (Dht, potent androgen) to 3␣/␤-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3␣/␤-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17␤-glucuronide (DhtG), Dht-17␤-sulfate (DhtS), and Tib-17␤-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 3␤-reduction (with DhtG) from 3␣-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 3␣-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. Aldo-ketoreductase (AKR)2 1C enzymes are cytosolic hydroxysteroid dehydrogenases (HSDs) that catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids (1). Four isoforms are known to exist in humans: AKR1C1 (20␣[3␣]-HSD), AKR1C2 (human 3␣-HSD type 3; also known as bile acid-binding protein), AKR1C3 (human 3␣-HSD type 2; also known as human 17␤-HSD type 5), and AKR1C4 (human 3␣-HSD type 1). Despite their high sequence identity (Ͼ86%), each individual human AKR1C enzyme displays distinct positional and stereochemical substrate preference and tissue distribution patterns. These enzymes are able to reduce 3-, 17-, and 20-ketosteroids and accept a broad spectrum of natural or synthetic steroids as substrates. They are implicated in diverse physiological functions by regulating the metabolism of androgens, estrogens, and progestins (1-3). Interestingly, distantly related human aldose reductase AKR1B1 shows superior turnover of glutathione conjugated lipid aldehydes than the unconjugated aldehydes (4). However, the ability of AKR1C isoforms to turnover steroid conjugates has not been previously examined.AKR1C isoforms are involved in the metabolism of 5␣-di...

Conversion of Human Steroid 5β-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H

“…In addition, the k cat /K m value for the reduction of 5␣-DHT catalyzed by the E120H mutant was about 15-fold greater than that observed for the reduction of this steroid to the opposite stereoisomer, 5␣-androstane-3␣,17␤-diol, catalyzed by AKR1C2. The values obtained with the mutant were similar to that observed for the reduction of 5␣-DHT to 5␣-androstane-3␣,17␤-diol catalyzed by AKR1C4, the most efficient human AKR1C enzyme (23).…”

“…enzymes reduce 5␣-DHT and 5␤-DHT with similar catalytic efficiencies (23,24). The preference of the AKR1D1 E120H mutant to reduce a 3-ketosteroid with an A/B trans-ring configuration over a cis-configuration by more than 10-fold was unexpected.…”

“…Comparison of AKR1D1 E120H Mutant with AKR1C Enzymes-AKR1C enzymes perform ketosteroid reduction and exhibit similar catalytic efficiencies for the reduction of 5␣-and 5␤-DHT and preferably form the 3␣-hydroxysteroid over the 3␤-hydroxysteroid (23,24). The exception is AKR1C1, which reduces 5␣-DHT to a mixture of 3␣-and 3␤-isomers in a 1:3 ratio.…”

Physiological Relevance of Pregnanolone Isomers and Their Polar Conjugates with Respect to the Gender, Menstrual Cycle and Pregnancy

“…On the other hand, AKR1Cs may decrease the neurosteroid concentrations by inactivating 3Ǐ,5Ǐ-THP and eliminating the precursors like progesterone from the synthetic pathways via reduction of the 20-oxo-steroid group (Penning, et al, 2000;Usami, et al, 2002). The AKR1C2 preferring 3Ǐ-reduction over the 3ǐ-reduction may catalyze 3Ǐ-, 17ǐ-, and 20Ǐ-HSD reactions (Jin, et al, 2009;Penning, et al, 2000;Usami, et al, 2002). From the family of short chain dehydrogenases (SDRs), type 7 17ǐ-hydroxysteroid dehydrogenase (HSD17B7), preferring the reduction of the oxo-groups in 20-, 17-or 3-position to the corresponding 20Ǐ-hydroxy-, 17ǐ-hydroxy-or 3Ǐ-hydroxy-counterparts, is also significantly expressed in the liver (Krazeisen, et al, 1999;Torn, et al, 2003).…”

“…Human liver contains various isoforms of pluripotent aldoketo reductases (AKR1C1, AKR1C2, AKR1C3, and AKR1C4) with 20Ǐ-, 17ǐ-, 3Ǐ-or 3ǐ-hydroxysteroid dehydrogenaselike activity (Jin, et al, 2009;Penning, et al, 2001;Shiraishi, et al, 1998). The enzyme activities could control the occupancy of GABA A -receptors (Penning, 1999) via reduction of oxo-groups in the steroid C3 position.…”