Mycobacterium tuberculosis exploits a heterohexameric enoyl-CoA hydratase retro-aldolase complex for cholesterol catabolism

Cholesterol catabolism plays an important role in Mycobacterium tuberculosis's (Mtb's) survival and persistence in the host. Mtb exploits three β-oxidation cycles to fully degrade the side-chain of cholesterol. Five cistronic genes in a single operon encode three enzymes, 3-oxo-4-pregnene-20-carboxyl-CoA dehydrogenase (ChsE1-ChsE2), 3-oxo-4,17-pregnadiene-20-carboxyl-CoA hydratase (ChsH1-ChsH2), and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA retro-aldolase (Ltp2) to perform the last β-oxidation cycle in this pathway. Among these three enzymes, ChsH1-ChsH2 and Ltp2 form a protein complex that is requisite for catalysis of carbon-carbon bond cleavage. In this work, we report the structure of full length ChsH1-ChsH2-Ltp2 complex based on small-angle X-ray scattering (SAXS) and single particle electron microscopy data. Mutagenesis experiments confirm the requirement for Ltp2 to catalyze the retro-aldol reaction. The structure illustrates how acyl transfer between enzymes may occur. Each protomer of ChsH1-ChsH2-Ltp2 contains three protein components: A chain of ChsH1, a chain of ChsH2, and a chain of Ltp2. Two protomers dimerize at the interface of Ltp2 to form a heterohexameric structure. This unique heterohexameric structure of ChsH1-ChsH2-Ltp2 provides entry to further understand the mechanism of cholesterol catabolism in Mtb.