NADP-dependent 3β-, 7α- and 7β-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c

Abstract

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3β-, 7α- and 7β-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3β-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3β-hydroxyl function could not be demonstrated. The lowest K m values were found for disubstituted bile acids, tribustituted and conjugated bile acids having higher K m values. 7α-Hydroxysteroid dehydrogenase, but not 7β-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells grown in the presence of chenodeoxycholic acid or 3α-hydroxy-7-keto-5β-cholanic acid. Ursodeoxycholic acid with its 7β-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82000 and 115000 were found for the 7α-hydroxysteroid dehydrogenase and the 7β-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive in vitro. Here, the pH optimum for the overall reaction was 8.5–8.7. 3β-, 7α- and 7β-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4°C, but were found to be heat-sensitive.