Bile acids are amphipathic steroid compounds produced in the digestive tract of vertebrates. After their excretion, these compounds can be used as carbon and energy sources by environmental bacteria. Degradation of bile acids proceeds via formation of Δ^1,4-intermediates in Actinobacteria and γ- and β-proteobacteria. In contrast, a pathway variant found in Sphingomonads such as Sphingobium sp. strain Chol11 involves formation of Δ^4,6-degradation intermediates from 7-hydroxylated bile acids. These intermediates are formed by water elimination from C7 catalyzed by the stereospecific 7α- and 7β hydroxysteroid dehydratases (Hsh2 and Hsh3, in strain Chol11) as key enzymes of this pathway variant.

The novel bile acid-degrading bacterium Novosphingobium sp. strain Chol12 also degrades 7-hydroxy bile acids via Δ^4,6-intermediates but exhibits significantly different kinetics of Δ^4,6 and Δ^1,4-intermediate production than strain Chol11. This study aimed to compare 7α- and 7β-hydroxysteroid dehydratase activities from strain Chol11 and strain Chol12 using C₂₄ and C₁₉ steroid substrates (with and without acyl side chain, respectively) with different hydroxylation patterns at C7 and C12. Enzyme assays with cell extracts from both strains presented similar 7α-dehydratase activity with C₁₉ substrates with a hydroxyl group in C12. In contrast, 7α-dehydratase activity was not observed in cell extracts of strain Chol12 when using the C12-hydroxylated substrate with C₂₄. This instance suggests that C12-hydroxyl group and the presence of an acyl side chain affect the Hsh2-like activity in strain Chol12. Assays for 7β-dehydratase activities of both strains are currently being carried out in our laboratory.

Genome sequencing of strain Chol12 revealed the presence of two proteins that share high sequence identity to Hsh2 and Hsh3 of Chol11. The function and substrate specificities of these homologs are currently studied at the biochemical level by heterologous expression and enzyme assays.

These results provide novel insights into the substrate specificity of hydroxysteroid dehydratases and might be relevant for the biotechnological production of pharmaceutical steroid compounds.