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 C 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. sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ 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-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 ( Chol1, CNB-2, and 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 resulted in a strong growth rate decrease with cholate, while growth with steroids with C side chains or without side chains was not affected. Intermediates with a 7-deoxy-3-keto-Δ 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 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. 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 CO-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.

The 7α‐hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α‐hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7

“…Bile salts and their degradation intermediates in culture supernatants were analysed by high‐performance liquid chromatography coupled to mass spectrometry (HPLC‐MS; (Yücel et al ., ) equipped with a reversed‐phase C 18 column (Knauer; 150 x 3 mm, Eurosphere II, 100‐5 C18) at 25 °C. Ammonium‐acetate buffer (10 mM, pH 6.7, eluent A) and acetonitrile (eluent B) were used as eluents with a flow rate of 0.3 ml min −1 .…”

“…Cultures were harvested in the midexponential growth phase by centrifugation at 8800 x g for 10 min at 4 C. Cell disruption was carried out as described for Novosphingobium sp. strain Chol11 (Yücel et al, 2018b). Protein concentrations were determined using the BCA assay (Pierce, Thermo Scientific, Rockford, IL) using bovine serum albumin as standard.…”

“…Bile salts and their degradation intermediates in culture supernatants were analysed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS; (Yücel et al, 2018b) equipped with a reversed-phase C 18 column (Knauer; 150 x 3 mm, Eurosphere II, 100-5 C18) at 25 C. Ammonium-acetate buffer (10 mM, pH 6.7, eluent A) and acetonitrile (eluent B) were used as eluents with a flow rate of 0.3 ml min −1 . The gradient HPLC method started with 10% eluent B for 2 min, increased to 48% eluent B within 25 min and returned to 10% eluent B within 1 min, followed by an equilibration of 5 min at a flow rate of 0.4 ml min −1 .…”

“…Ionization of samples and mass spectrometric detection was achieved as described previously (Yücel et al, 2018b).…”

“…In strain Novosphingobium sp. strain Chol11 HOCDA is further degraded to 12βhydroxy-androsta-1,4,6-triene-3,17-dione (HATD) via sofar unknown reactions initiated by an acyl-CoA ligase (Yücel et al, 2018b).…”