Abstract
25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3α-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12α -hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12α -hydroxysteroid dehydrogenase also contained 3α-hydroxysteroid dehydrogenase although 12α-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3α-hydroxysteroid dehydrogenase. In addition, 7α-hydroxysteroid dehydrogenase activity was evident only when 3α,7α,12α-trihydroxy-5β-cholanoate was substrate but notably absent when 3α,7α-dihydroxy-5β-cholanoate was substrate. The oxidation product 12α-hydroxy-3,7-diketo-5β-cholanoate is rapidly further degraded to an unknown compound devoid of either 3α- or 7α-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens, Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3α-OH- and 12α-OH-oriented activities, respectively. Kinetic studies gave K m estimates of approx. 5 × 10 −5 and 8 × 10 −4 M with 3α, 7α-dihydroxy-5β-cholanoate and 3α,12α-dihydroxy-5β-cholanoate as substrates for the two respective enzymes. 3α-hydroxysteroid dehydrogenase was active against 3α-OH-containing steroids such as androsterone regardless of the stereochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3β-OH-containing steroids. The 3α- and 12α-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.
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