Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.
A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.
D001345:Autoradiography; D002855:Chromatography, Thin Layer; D003900:Desoxycorticosterone / Q000378:metabolism; D005051:Eubacterium / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D005182:Flavin-Adenine Dinucleotide; D003900:Desoxycorticosterone; D009249:NADP; D005486:Flavin Mononucleotide; D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase; D009252:NADPH Dehydrogenase