Biochemical and Biophysical Research Communications, Vol.272, No.2, 622-628, 2000
Functional expression, purification, and characterization of 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni
3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO2 and H2O in Comamonas testosteroni. By this activity, 3 alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3 alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5 alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha-HSD/CR). It is suggested that 3 alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3 alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3 alpha-HSD/CR of C. testosteroni.