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


ABSTRACT: Mycobacterium tuberculosis (Mtb) 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 (R)-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 (kcat/Km = 5.8 ± 0.8 × 104 M-1?s-1). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a ?-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 ?-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105A, conserved in the ?-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB·COCHEA-CoA complex, solved to 1.4 Å, revealed that Glu105A is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105AA variant accumulated a yellow-colored species (?max = 310 nm; Kd = 0.4 ± 0.2 ?M) typical of ?-keto enolates. In the presence of D2O, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with kcat 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 Mtb pathogenesis.

SUBMITTER: Crowe AM 

PROVIDER: S-EPMC5899439 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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IpdAB, a virulence factor in <i>Mycobacterium tuberculosis</i>, is a cholesterol ring-cleaving hydrolase.

Crowe Adam M AM   Workman Sean D SD   Watanabe Nobuhiko N   Worrall Liam J LJ   Strynadka Natalie C J NCJ   Eltis Lindsay D LD  

Proceedings of the National Academy of Sciences of the United States of America 20180326 15


<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 (COCHE  ...[more]

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