Characterization of 3,17β-hydroxysteroid dehydrogenase in Comamonas testosteroni.

Abstract 3,17β-Hydroxysteroid dehydrogenases (3,17β-HSDs) are found in all forms of life which catalyze the 3-position and 17-position reduction/oxidation of steroids. Comamonas testosteroni ( C. testosteroni ) ATCC11996 is a gram-negative bacterium which can use steroids as carbon and energy source. 3,17β-HSD is an enzyme which is involved in the complete oxidative degradation of the steroid skeleton, induced in the presence of these compounds in the culture medium. Cyclizing RT-PCR (cRT-PCR) was used to investigate the transcription start site (TSS) and promoter of 3,17β-HSD gene. To prove that 3,17β-HSD is involved in the metabolic pathway of steroid compounds, we prepared a 3,17β-HSD gene knock-out mutant and a mutant of C. testosteroni in which 3,17β-HSD was expressed at high level. The results indicate that 3,17β-HSD gene expression was induced by testosterone, but not by estradiol and cholesterol. Compared to the wild type C. testosteroni , degradation ability of testosterone and cholesterol was almost lost, and degradation of estradiol was decreased in the 3,17β-HSD knock-out mutant. Meanwhile degradation of testosterone, cholesterol was obviously increased in the 3,17β-HSD high expression mutant. Furthermore, the growths in the medium with testosterone, cholesterol or estradiol were impaired in 3,17β-HSD knock-out mutant. The results showed that in addition to testosterone and estradiol, 3,17β-HSD might be also involved in cholesterol metabolism. The location of the TSS and promoter of the 3,17β-HSD gene were found in this work.