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A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts.

ABSTRACT: Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-?4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7?-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-seco degradation pathway used by other steroid-degrading bacteria indicating that strain Chol11 uses an alternative pathway. A reciprocal BLASTp analysis showed that known side chain degradation genes from other cholate-degrading bacteria (Pseudomonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1) were not found in the genome of strain Chol11. The characterization of a transposon mutant of strain Chol11 showing altered growth with cholate identified a novel steroid-24-oyl-coenzyme A ligase named SclA. The unmarked deletion of sclA resulted in a strong growth rate decrease with cholate, while growth with steroids with C3 side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-?4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid (DOCDA), were shown to be likely physiological substrates of SclA. Furthermore, a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with an additional double bond in the side chain was identified. These results support the hypothesis that Novosphingobium sp. strain Chol11 harbors an alternative pathway for cholate degradation, in which side chain degradation is initiated by the CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknown enzymes different from those of other steroid-degrading bacteria.IMPORTANCE This study provides further evidence of the diversity of metabolic pathways for the degradation of steroid compounds in environmental bacteria. The knowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating the fate of pharmaceutical steroids in the environment, some of which may act as endocrine disruptors.

SUBMITTER: Yucel O

PROVIDER: S-EPMC5734017 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts.

Yücel Onur O Holert Johannes J Ludwig Kevin Christopher KC Thierbach Sven S Philipp Bodo B

Applied and environmental microbiology 20171215 1

Bile salts such as cholate are steroid compounds with a C5 carboxylic side chain and occur ubiquitously in vertebrates. Upon their excretion into soils and waters, bile salts can serve as growth substrates for diverse bacteria. Novosphingobium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabolites by the dehydration of the 7-hydroxyl group catalyzed by the 7α-hydroxysteroid dehydratase Hsh2. This reaction has not been observed in the well ...[more]

PMID: 29054875

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Project description:The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ4,6-variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations of strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase (ACAD) Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ22-ACAD Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts, while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δscd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ4,6-steroids, while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228, led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of proteobacterial enzymes for bile-salt degradation and suggest a novel side chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with nonnative substrates.

Project description:UnlabelledThe acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of Mycobacterium tuberculosis and Rhodococcus jostii RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the casC gene in R. jostii RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.ImportanceWe report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from M. tuberculosis and Rhodococcus jostii RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogen M. tuberculosis within host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.

Dataset Information

A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts.

Publications

A Novel Steroid-Coenzyme A Ligase from Novosphingobium sp. Strain Chol11 Is Essential for an Alternative Degradation Pathway for Bile Salts.

Similar Datasets

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