Actinobacterial acyl coenzyme A synthetases involved in steroid side-chain catabolism

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 (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 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 (kcat/Km = 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.

FIG 1

Chemical structure of cholesterol. Rings are labeled A to D, and atoms are numbered 1 to 27, according to IUPAC-recommended nomenclature.

FIG 2

Reactions catalyzed by acyl-CoA synthetases.

FIG 3

Dendrogram showing the phylogeny of bacterial acyl-CoA synthetases. Coding sequences are identified by name or number and the strain in which they occur. CNB2, Comamonas testosteroni CNB-2; TA441, Comamonas testosteroni TA441; Gbro, Gordonia bronchialis DSM 43247; KTR9, Gordonia sp. strain KTR9; HNE, Hyphomonas neptunium ATCC 15444; Mps, Mycobacterium parascrofulaceum ATCC BAA-614; MSMEG, Mycobacterium smegmatis MC²155; Mtb, Mycobacterium tuberculosis H37Rv; MYSTI, Myxococcus stipitatus DSM 14675; nfa, Nocardia farcinica IFM 10152; Chol1, Pseudomonas sp. strain Chol1; DOC21, Pseudomonas putida DOC21; P1, Pseudonocardia sp. strain P1; PSHA, Pseudoalteromonas haloplanktis TAC125; RHA1, Rhodococcus jostii RHA1; REQ, Rhodococcus equi 103S; RER, Rhodococcus erythropolis PR4; SACE, Saccharopolyspora erythraea NRRL 2338; Strop, Salinispora tropica CNB-440; Sulb, Sulfitobacter sp. strain EE-36; Tcur, Thermomonospora curvata DSM 43183; BaiB, bile acid:CoA ligase. The conservation of the C-terminal PX₄GK motif required for acetylation is represented on the tree by an “a” in superscript. Dark- and light-shaded groups contain only actinobacterial and proteobacterial sequences, respectively.

FIG 4

Steady-state kinetic analysis of CasG_RHA1 for cholate. Shown is the dependence of the initial velocity (V_o) of AMP formation on the cholate concentration. The solid line represents a best fit of the nonhyperbolic substrate inhibition equation to the data (K_S = 160 ± 20 μM; V_max = 14 ± 1 μM min⁻¹; K_i = 1,200 ± 300 μM; R² ≥ 0.97).