Proteome, Bioinformatic, and Functional Analyses Reveal a Distinct and Conserved Metabolic Pathway for Bile Salt Degradation in the Sphingomonadaceae.

Bile salts are amphiphilic steroids with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile salt steroid skeleton resembles the well-studied pathway for other steroids, like testosterone, while specific differences occur during side chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ1,4- or Δ4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ1,4 variant is well known from many model organisms, the Δ4,6 variant involving a 7-hydroxysteroid dehydratase as a key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic, and functional analyses of the Δ4,6 variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to that of the Δ1,4 variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for the degradation of the C5 carboxylic side chain encoding a CoA ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ4,6 variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side chain degradation cluster. A second amidase in the side chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile salt hydrolases. This study identifies members of the Sphingomonadaceae that are remarkably well adapted to the utilization of bile salts via a partially distinct metabolic pathway. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

Substrate Inhibition of 5ß-Δ4-3-Ketosteroid Dehydrogenase in Sphingobium sp. Strain Chol11 Acts as Circuit Breaker During Growth With Toxic Bile Salts.

In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5ß-steroids, which leads to a bent structure of the steroid core. Bile salts are surface-active steroids excreted into the environment in large amounts, where they are subject to bacterial degradation. Bacterial steroid degradation is initiated by the oxidation of the A-ring leading to canonical Δ4-3-keto steroids with a double bond in the A-ring. For 5ß-bile salts, this Δ4-double bond is introduced into 3-keto-bile salts by a 5ß-Δ4-ketosteroid dehydrogenase (5ß-Δ4-KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5ß-Δ4-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5ß-Δ4-KSTD1. By heterologous production in Escherichia coli, 5ß-Δ4-KSTD function could be shown for 5ß-Δ4-KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5ß-Δ4-kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5ß-Δ4-KSTD1, showed delayed 3-ketocholate transformation. Purified 5ß-Δ4-KSTD1 was specific for 5ß-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5ß-Δ4-KSTD1 showed a relatively low K m for 3-ketocholate, a very high specific activity and pronounced substrate inhibition. With respect to the toxicity of bile salts, these kinetic properties indicate that 5ß-Δ4-KSTD1 can achieve fast detoxification of the detergent character as well as prevention of an overflow of the catabolic pathway in presence of increased bile-salt concentrations.