Publications

• Catabolism of the last two steroid rings in Mycobacterium tuberculosis and other bacteria.

  Crowe A.M., Casabon I., Brown K.L., Liu J., Lian J., Rogalski J.C., Hurst T.E., Snieckus V., Foster L.J., Eltis L.D.

  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 <i>Mycobacterium tuberculosis</i>, this pathway includes virulence determinants and has been proposed to be encoded b ... >> More

  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 <i>Mycobacterium tuberculosis</i>, 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 (<i>ipdAB</i>) and an acyl-CoA reductase gene (<i>ipdC</i>). In the presence of cholesterol, Δ<i>ipdC</i> and Δ<i>ipdAB</i> mutants of either <i>M. tuberculosis</i> or <i>Rhodococcus jostii</i> strain RHA1 accumulated previously undescribed metabolites: 3aα-<i>H</i>-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (<i>R</i>)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A Δ<i>fadE32</i> mutant of <i>Mycobacterium smegmatis</i> 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<i>ipdF</i> and Δ<i>echA20</i> 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<i>ipdAB M. tuberculosis</i> 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 <i>M. tuberculosis.</i><b>IMPORTANCE</b> Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In <i>Mycobacterium tuberculosis</i>, this pathway includes virulence determinants. Using a series of mutants in <i>M. tuberculosis</i> 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. << Less

  MBio 8:e00321-e00321(2017) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.

  Crowe A.M., Workman S.D., Watanabe N., Worrall L.J., Strynadka N.C.J., Eltis L.D.

  <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs ... >> More

  <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) grows on host-derived cholesterol during infection. IpdAB, found in all steroid-degrading bacteria and a determinant of pathogenicity, has been implicated in the hydrolysis of the last steroid ring. Phylogenetic analyses revealed that IpdAB orthologs form a clade of CoA transferases (CoTs). In a coupled assay with a thiolase, IpdAB transformed the cholesterol catabolite (<i>R</i>)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) and CoASH to 4-methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) and acetyl-CoA with high specificity (<i>k</i>_cat/<i>K</i>_m = 5.8 ± 0.8 × 10⁴ M^-1⋅s^-1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a β-keto-thioester, a thiolase substrate. Contrary to characterized CoTs, IpdAB exhibited no activity toward small CoA thioesters. Further, IpdAB lacks the catalytic glutamate residue that is conserved in the β-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105^A, conserved in the α-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 Å, revealed that Glu105^A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105A^A variant accumulated a yellow-colored species (λ_max = 310 nm; <i>K</i>_d = 0.4 ± 0.2 μM) typical of β-keto enolates. In the presence of D₂O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with <i>k</i>_cat Based on these data and additional IpdAB variants, we propose a retro-Claisen condensation-like mechanism for the IpdAB-mediated hydrolysis of COCHEA-CoA. This study expands the range of known reactions catalyzed by the CoT superfamily and provides mechanistic insight into an important determinant of <i>Mtb</i> pathogenesis. << Less

  Proc. Natl. Acad. Sci. U.S.A. 115:E3378-E3387(2018) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.