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

Abstract

<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.