AUTHOR=Feller Franziska M. , Marke Gina , Drees Steffen L. , Wöhlbrand Lars , Rabus Ralf , Philipp Bodo TITLE=Substrate Inhibition of 5β-Δ4-3-Ketosteroid Dehydrogenase in Sphingobium sp. Strain Chol11 Acts as Circuit Breaker During Growth With Toxic Bile Salts JOURNAL=Frontiers in Microbiology VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.655312 DOI=10.3389/fmicb.2021.655312 ISSN=1664-302X ABSTRACT=

In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5β-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 5β-bile salts, this Δ⁴-double bond is introduced into 3-keto-bile salts by a 5β-Δ⁴-ketosteroid dehydrogenase (5β-Δ⁴-KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5β-Δ⁴-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5β-Δ⁴-KSTD1. By heterologous production in Escherichia coli, 5β-Δ⁴-KSTD function could be shown for 5β-Δ⁴-KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5β-Δ⁴-kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5β-Δ⁴-KSTD1, showed delayed 3-ketocholate transformation. Purified 5β-Δ⁴-KSTD1 was specific for 5β-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5β-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 5β-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.