Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

Abstract

ABSTRACT Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis . Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19 Mtb ) transformed 3-oxo-4-cholesten-26-oate ( k cat / K m = 0.33 × 10 5 ± 0.03 × 10 5 M −1 s −1 ) and represents orthologs that activate the C 8 side chain of cholesterol. Both CasG RHA1 and FadD17 Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C 5 side chain of cholate, while FadD17 and its orthologs appear to activate the C 5 side chain of one or more cholesterol metabolites. CasI RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C 3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα- H -4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone ( k cat /K m = 2.4 × 10 5 ± 0.1 × 10 5 M −1 s −1 and 3.2 × 10 5 ± 0.3 × 10 5 M −1 s −1 , respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria . Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.