Identification and characterization of a 20β-HSDH from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058

摘要

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20{beta}-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20{beta}-HSDH in both B. desmolans and C. cadaveris. The 20{beta}-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 {+/-} 3.7 kDa. The r20{beta}-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0-7.5 with (20{beta}-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with Km, 0.80 {+/-} 0.06 M; Vmax, 30.36 {+/-} 1.97 mol{middle dot}min-1; Kcat, 607 {+/-} 39 mol{middle dot}M-1{middle dot}min-1; Kcat/Km, 760 {+/-} 7.67. Phylogenetic analysis of the 20{beta}-HSDH from B. desmolans suggested that the 20{beta}-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20{beta}-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.