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2021
Journal Article
Title
Substrate Inhibition of 5v-D4-3-Ketosteroid Dehydrogenase in Sphingobium sp. Strain Chol11 Acts as Circuit Breaker During Growth With Toxic Bile Salts
Abstract
In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5v-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 D4-3-keto steroids with a double bond in the A-ring. For 5v-bile salts, this D4-double bond is introduced into 3-keto-bile salts by a 5v-D4-ketosteroid dehydrogenase (5v-D4-KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5v-D4-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5v-D4-KSTD1. By heterologous production in Escherichia coli, 5v-D4-KSTD function could be shown for 5v-D4-KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5v-D4-kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5v-D4-KSTD1, showed delayed 3-ketocholate transformation. Purified 5v-D4-KSTD1 was specific for 5v-steroids in contrast to 5a-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5v-D4-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 5v-D4-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.