Steroid 5alpha-reductase activity of Treponema denticola.
BACKGROUND
Previously we have shown that reference and freshly isolated Treponema denticola cultures possess 5alpha-reductase (5alpha-R) and 3beta- and 17beta-hydroxysteroid dehydrogenase activity. A gene matching the 3-oxo-5alpha-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 5alpha-R activity of T. denticola ATCC 33520.


METHODS
5alpha-R activity of cell-free preparations was assayed with radioactive steroid substrates. 5alpha-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 5alpha-R was 5.5 and the preferred co-factor was NADPH. The order of the steroids with respect to their 5alpha-R substrate specificities was (in descending order): progesterone, 4-androstenedione, testosterone and corticosterone. There are at least two intermediates in the synthesis of 5alpha-dihydrocholesterol from cholesterol.


CONCLUSIONS
These results suggest that the 3-oxo-5alpha-steroid 4-dehydrogenase family protein gene of T. denticola codes for a functional protein that resembles mammalian 5alpha-R isoenzyme 2 with regard to co-factor requirement and pH optimum.
Clark
D T
DT
Department of Microbiology, King's College London Dental Institute at Guy's, King's College and St Thomas' Hospitals, London, UK. douglas.2.clark@kcl.ac.uk
Soory
M
M
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D002021:Buffers; D002250:Carbon Radioisotopes; D004083:Cholestanol / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003067:Coenzymes; D003345:Corticosterone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009249:NADP / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D019275:Radiopharmaceuticals; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D013997:Time Factors; D048373:Treponema denticola / Q000201:enzymology*