Characterization of an NADPH-dependent 17ɑ-hydroxysteroid dehydrogenase encoded by the desF gene from the gut...

Epitestosterone (epiT) is the isomer of the androgen testosterone. Historically, the role of epiT has remained unclear. Recently, it has been reported that epiT promotes nuclear androgen receptor (AR)-dependent prostate cancer cell proliferation. The gut bacterium Clostridium scindens VPI 12708 was shown to convert androstenedione (AD) to epiT over three decades ago. The bacterial enzymatic pathways involved in epiT formation have only recently been reported. The desF gene encodes 17α-hydroxysteroid dehydrogenase which converts AD to epiT using NADPH as a cofactor. In this study, we quantitatively characterized DesF kinetic parameters and substrate specificity. The results revealed that the optimal pH for the reductive reaction is 7.0, and for the oxidative reaction it is 7.5 and 8.0. The kinetic analysis showed that for the reductive reaction, the K_M was 8.1 ± 1.8 µM and the V_max was 6.4 ± 0.3 µmol·min^-1·mg^-1; for the oxidative direction, the K_M was 27.3 ± 3.3 µM and the V_max was 7.2 ± 0.3 µmol·min^-1·mg^-1. Moreover, the substrate specificity analysis revealed that 11-keto-AD is the most favourable substrate for DesF, and the 17-keto group of 11-keto-AD can be converted to the 17α-hydroxy group. The phylogenetic relation between DesF and other characterized hydroxysteroid dehydrogenases reveals common ancestry with human HSD17B10 and Eggerthella lenta 3β-HSDH. These results are a significant advance in understanding epiT formation by the gut microbiome.