Catabolism of the Last Two Steroid Rings in and Other Bacteria

Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In , this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene () and an acyl-CoA reductase gene (). In the presence of cholesterol, Δ and Δ mutants of either or strain RHA1 accumulated previously undescribed metabolites: 3aα--4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and ()-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A Δ mutant of accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by Δ and Δ mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a Δ mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In , this pathway includes virulence determinants. Using a series of mutants in and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics.