Human dehydrogenase/reductase SDR family member 11 (DHRS11) and aldo-keto reductase 1C isoforms in comparison: Substrate and reaction specificity in the reduction of 11-keto-C19-steroids

Recent studies have shown that an adrenal steroid 11 beta-hydroxy-4-androstene-3,17-dione serves as the precursor to androgens, 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT). The biosynthetic pathways include the reduction of 3- and 17-keto groups of the androgen precursors 11-keto-C-19-steroids, which has been reported to be mediated by three human enzymes; aldo-keto reductase (AKR)1C2, AKR1C3 and 17 beta-hydroxysteroid dehydrogenase (HSD) type-3. To explore the contribution of the enzymes in the reductive metabolism, we kinetically compared the substrate specificity for 11-keto-C-19-steroids among purified recombinant preparations of four AKRs (1C1, 1C2,1C3 and 1C4) and DHRS11, which shows 17 beta-HSD activity. Although AKR1C1 did not reduce the 11-keto-C-19-steroids, AKR1C3 and DHRS11 reduced 17-keto groups of 11-keto-4-androstene-3,17-dione, 11-keto-5 alpha-androstane-3,17-dione (11K-Adione) and 11-ketoandrosterone with K-m values of 5-28 mu M. The 3-keto groups of 11KDHT and 11K-Adione were reduced by AKR1C4 (K-m 1 mu M) more efficiently than by AKR1C2 (K-m 5 and 8 mu M, respectively). GC/MS analysis of the products showed that DHRS11 acts as 17 beta-HSD, and that AKR1C2 and AKR1C4 are predominantly 3 alpha-HSDs, but formed a minor 3 beta-metabolite from 11KDHT. Since DHRS11 was thus newly identified as 11-keto-C-19-steroid reductase, we also investigated its substrate-binding mode by molecular docking and site-directed mutagenesis of Thr163 and Val200, and found the following structural features: 1). There is a space that accommodates the 11-keto group of the 11-keto-C-19-steroids in the substrate-binding site. 2) Val200 is a critical determinant for exhibiting the strict 17 beta-HSD activity of the enzyme, because the Val200Leu mutation resulted in both significant impairment of the 17 beta-HSD activity and emergence of 3 beta-HSD activity towards 5 alpha-androstanes including 11KDHT.