Bile acids are steroids produced by vertebrates in their digestive tract, and after excretion, they can be used as carbon and energy sources by environmental bacteria. Bile acids have a hydroxylated steroid skeleton with a C₅ side chain attached to C17. Two pathway variants are known for bile acid catabolism under aerobic conditions, named Δ^1,4 and Δ^4,6 after the unsaturation of their respective intermediates. While the Δ^1,4-variant is common in pseudomonads and Actinobacteria, the Δ^4,6-variant seems unique to the Sphingomonadaceae family.

Proteomic analysis of Sphingobium sp. strain Chol11 grown with the bile acid cholate revealed a gene cluster specifically upregulated during side-chain degradation. It contains genes for a CoA ligase (sclA), an acyl-CoA-dehydrogenase (scd4AB), an amidase, and the putative Rieske Monooxygenase (RMO) gene nov2c228. Notably, this cluster lacks genes for stepwise side-chain degradation, like thiolases and aldolases as described in organisms having the Δ^1,4-variant such as Pseudomonas stutzeri Chol1. Additionally, strain Chol11 Δscd4A cannot grow with cholate and accumulates hydroxylated metabolites with a C₅ side-chain. These instances suggest that side-chain degradation in strain Chol11 involves a hydroxylation catalyzed by Nov2c228.

The potential role of the RMO Nov2c228 was approached by gene deletion and heterologous expression. Attempts to delete the gene resulted in the apparent loss of the whole cluster and have been, thus, inconclusive. Heterologous expression in P. stutzeri Chol1 ΔstdA1 ΔkstD cells, which cannot degrade bile acids with a C₅ side-chain, showed that the Δ^4,6-compound 3-oxo-chol-4,6-dienoate (OCDA), a presumptive Nov2c228 substrate, was converted into a compound 16 Da heavier, suggesting that a hydroxylation took place. Ongoing studies aim to characterize this hydroxylation, including the structure of the compound.

This study provides further evidence that bile acid side-chain degradation in Sphingomonadaceae proceeds via an unknown pathway involving an RMO-catalyzed hydroxylation.