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Publication Abstract from PubMed

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 x 10(4) M(-1)s(-1)). The structure of MOODA-CoA was consistent with IpdAB hydrolyzing COCHEA-CoA to a beta-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 beta-subunit of characterized CoTs and a glutamyl-CoA intermediate was not trapped during turnover. By contrast, Glu105(A), conserved in the alpha-subunit of IpdAB, was essential for catalysis. A crystal structure of the IpdAB.COCHEA-CoA complex, solved to 1.4 A, 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 (lambdamax = 310 nm; Kd = 0.4 +/- 0.2 muM) typical of beta-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.

IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.,Crowe AM, Workman SD, Watanabe N, Worrall LJ, Strynadka NCJ, Eltis LD Proc Natl Acad Sci U S A. 2018 Mar 26. pii: 1717015115. doi:, 10.1073/pnas.1717015115. PMID:29581275^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.