Investigations on bile acid catabolism in Sphingobium sp. strain Chol11 suggests a Rieske monooxygenase-catalyzed hydroxylation that occurs prior to the degradation of the carboxylic side chain

Bile acids are steroids produced by vertebrates in their digestive system, 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 carboxylic side chain attached to C17. Two pathway variants, named Δ^1,4 and Δ^4,6, are known for bile acid catabolism under aerobic conditions. 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 bile acids revealed a gene cluster specifically upregulated during side-chain degradation, which is conserved in this family. It contains genes for a CoA ligase (sclA), an acyl-CoA-dehydrogenase (scd4AB), as well as genes for an amidase and a Rieske monooxygenase (RMO). Notably, this cluster lacks genes for the stepwise side-chain degradation described in bacteria using the Δ^1,4 variant. Additionally, Chol11 Δscd4A cannot grow with bile acids and accumulates hydroxylated dead-end metabolites with a C₅ side-chain. These instances suggest that side-chain degradation in strain Chol11 involves a hydroxylation catalyzed by the RMO.

The potential role of the RMO was investigated through gene deletion and complementation experiments. Gene deletion attempts led to the removal of a 64 kb genomic region, encompassing a substantial fraction of the side-chain degradation cluster containing the genes coding for the RMO, the amidase, and the acyl-CoA dehydrogenase Scd4AB, plus a downstream genomic region. Complementation of this mutant with the genes coding for RMO only, as well as with the amidase and RMO genes together, resulted in the production of hydroxylated metabolites containing an intact side chain. These metabolites resembled the dead-end metabolite production by strain Chol11 Δscd4A. These results indicate that RMO catalyzes the hydroxylation of intermediates in the Δscd4A mutant and suggest that these hydroxylated compounds might be the substrate for Scd4AB before the side-chain degradation.

This study provides further evidence that bile acid side-chain degradation in Sphingomonadaceae proceeds via an uncharacterized pathway involving an RMO-catalyzed hydroxylation. Current efforts are focused on complementing strain Chol11 m25 with different gene combinations comprising scd4AB, RMO, and amidase genes in order to elucidate the putative reaction sequence catalyzed by the respective enzymes.