6COJ

Crystal structure of Rhodococcus jostii RHA1 IpdAB E105A COCHEA-COA complex

Summary for 6COJ

• Entry DOI: 10.2210/pdb6coj/pdb
• Descriptor: Probable CoA-transferase alpha subunit, Probable CoA-transferase beta subunit, SULFATE ION, ... (5 entities in total)
• Functional Keywords: cholesterol, ring cleaving, virulence factor, hydrolase
• Biological source: Rhodococcus jostii (strain RHA1); More
• Total number of polymer chains: 2
• Total formula weight: 62537.16

Authors

Crowe, A.M.,Workman, S.D.,Watanabe, N.,Worrall, L.J.,Strynadka, N.C.J.,Eltis, L.D. (deposition date: 2018-03-12, release date: 2018-03-28, Last modification date: 2023-10-04)

Primary citation

Crowe, A.M.,Workman, S.D.,Watanabe, N.,Worrall, L.J.,Strynadka, N.C.J.,Eltis, L.D.
IpdAB, a virulence factor inMycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.
Proc. Natl. Acad. Sci. U.S.A., 115:E3378-E3387, 2018

PubMed Abstract: () 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 ()-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 (/ = 5.8 ± 0.8 × 10 M⋅s). 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, 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 is positioned to act as a catalytic base. Upon titration with COCHEA-CoA, the E105A variant accumulated a yellow-colored species (λ = 310 nm; = 0.4 ± 0.2 μM) typical of β-keto enolates. In the presence of DO, IpdAB catalyzed the deuteration of COCHEA-CoA adjacent to the hydroxylation site at rates consistent with 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 pathogenesis.

PubMed: 29581275
DOI: 10.1073/pnas.1717015115

Experimental method

X-RAY DIFFRACTION (1.4 Å)