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

Franziska M Feller,Lars Wöhlbrand,Steffen L Drees,Ralf Rabus,Bodo Philipp,Gina Marke

doi:10.3389/fmicb.2021.655312

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 Km 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.

Highlights

Bile salts such as cholate (1 in Figure 1) are an important class of steroid compounds in vertebrates that aid in digestion of fatty compounds of food (Hofmann et al, 2010)
Whereas 3-ketobile salt oxidation was tested in bile-salt degrading strains Sphingobium sp. strain Chol11, P. stutzeri Chol1 and R. jostii RHA1, E. coli MG1655 was used for heterologous expression of untagged putative 5β-Δ4-Ketosteroid dehydrogenase (KSTD) and E. coli Tuner was used for production of his-tagged 5β-Δ4-KSTD for purification. 5-aminolevulinic-acid auxotrophic strain E. coli ST18 was used for transferring plasmids to the other strains by biparental conjugation
Wild type strains of P. stutzeri Chol1, Sphingobium sp. strain Chol11 and R. jostii RHA1 were grown with 1 mM cholate as carbon source, whereas deletion mutants of Sphingobium sp. strain Chol11 and E. coli MG1655 were grown with 15 mM glucose, and deletion mutants of P. stutzeri Chol1 as well as P. putida KT2440 were grown with 12 mM succinate

Summary

Introduction

Bile salts such as cholate (1 in Figure 1) are an important class of steroid compounds in vertebrates that aid in digestion of fatty compounds of food (Hofmann et al, 2010). Bile salts have a C5 carboxylic side chain attached to C17 of the steroid skeleton and a hydroxyl group at C3; many bile salts have one or two additional hydroxyl groups attached to C-atoms 6, 7, or 12, B. Large amounts of bile salts are released into the environment, for example, up to about 0.4–0.6 g bile salts per day by each human (Ridlon et al, 2006). Strain Chol (Philipp et al, 2006; Philipp, 2011; Horinouchi et al, 2012; Mohn et al, 2012; Holert et al, 2014; Bergstrand et al, 2016) Bile salts are carbon‐ and electron-rich substrates for heterotrophic bacteria such as Rhodococcus jostii RHA1, Pseudomonas stutzeri Chol as well as Sphingobium sp. strain Chol, formerly Novosphingobium sp. strain Chol (Philipp et al, 2006; Philipp, 2011; Horinouchi et al, 2012; Mohn et al, 2012; Holert et al, 2014; Bergstrand et al, 2016).

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