CHARACTERIZATION OF A 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE CARBONYL REDUCTASE FROM THE GRAM-NEGATIVE BACTERIUM COMAMONAS-TESTOSTERONI

A new form of the NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenas es (3 alpha-HSDs), present in the gram-negative bacterium Comamonas te stosteroni ATCC 11996, was isolated from a testosterone-induced bacter ial extract and characterized. The enzyme (HSD 28) has a monomeric mol ecular mass of 28 kDa. It belongs to the protein superfamily of short- chain dehydrogenases/reductases (SDR) as established by N-terminal seq uence analysis. Along with the 3 alpha-hydroxysteroid dehydrogenase an d 3-oxo-reductase activities towards a variety of cis or trans fused A /B ring steroids, it also reduces several xenobiotic carbonyl compound s, including a metyrapone-based class of insecticides, to the respecti ve alcohol metabolites. No dihydrodiol dehydrogenase activity towards trans- or cis-benzene-dihydrodiols could be detected, thus distinguish ing it from the indomethacine-sensitive, mammalian liver type 3 alpha- HSDs. Subcellular fractionation revealed that the enzyme is localized in the cytoplasm of the bacterial cell. Proteins similar to the 3 alph a-HSD were detected and characterized from Comamonas testosteroni stra in ATCC 17454 and from a commercially available steroid-induced extrac t of a patent Pseudomonas, strain. The N-terminal amino acid sequence of the 3 alpha-HSD from the latter strain (HSD 29) is highly similar ( 94% identity over 15 residues) to a previously determined primary stru cture of a Pseudomonas species 3 alpha-HSD. However, no similarities c ould be detected between HSD 28 and a recently determined 3 alpha-HSD sequence from the ATCC 11996 Comamonas, strain. The specific crossreac tion of antibodies directed against mammalian liver type I 11 beta-hyd roxysteroid dehydrogenase (11 beta-HSD I) with the isolated 3 alpha-HS Ds suggests the existence of a functionally and structurally related s ubgroup within the SDR superfamily. The broad substrate specificities of the characterized 3 alpha-HSD enzymes lead to the conclusion that t hey might participate in the intestinal bioactivation or inactivation of hormones, bile acids and xenobiotics since Comamonas testosteroni a nd related species are found in the intestinal tract of vertebrates in cluding man.