Steroid 5 alpha-reductase activity of Treponema denticola

Introduction: Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5 alpha-reductase (5 alpha-R) and 3 beta- and 17 beta-hydroxysteroid dehydrogenase activity. A gene matching the 3-oxo-5 alpha-steroid 4-dehydrogenase family protein (gene ID: 2739284; locus tag: TDE2697) has been identified in T. denticola ATCC 35405. The aim of the work presented here was to optimize assay conditions and determine steroid substrate specificities for the 5 alpha-R activity of T. denticola ATCC 33520. Methods: 5 alpha-R activity of cell-free preparations was assayed with radioactive steroid substrates. 5 alpha-R-reduced products were identified using thin-layer chromatography and a radioisotope scanner. Assay conditions were optimized for co-factor, buffer and pH requirements. Apparent substrate specificities were determined for progesterone, 4-androstenedione, testosterone and corticosterone. The time-course for metabolism of radiolabelled progesterone and cholesterol substrates was investigated with anaerobic cultures. Results: The optimum pH for 5 alpha-R was 5.5 and the preferred co-factor was NADPH. The order of the steroids with respect to their 5 alpha-R substrate specificities was (in descending order): progesterone, 4-androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5 alpha-dihydrocholesterol from cholesterol. Conclusion: These results suggest that the 3-oxo-5 alpha-steroid 4-dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5 alpha-R isoenzyme 2 with regard to co-factor requirement and pH optimum