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The catabolism of the last two rings of cholesterol by Mycobacterium tuberculosis

University of British Columbia

Cholesterol, a four-ringed steroid with an alkyl side chain, is an important growth substrate for Mycobacterium tuberculosis (Mtb) during infection. Many aspects of this catabolism remain unknown although steroid catabolism is a defining feature of mycobacteria and the related rhodococci. Using a variety of approaches, I elucidated key aspects of cholesterol catabolism in Mtb and other bacteria, particularly with respect to 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indane-dione (HIP), a metabolite that contains the last two steroid rings (C/D). Chapter 2 demonstrates that the first two steroid rings (A/B) are degraded prior to the side chain in mycobacteria and rhodococci. This was established by targeting HsaD, the final ings A/B-degrading enzyme. Thus, a ΔhsaD mutant of Rhodococcus jostii RHA1 accumulated cholesterol-derived catabolites with partially degraded side-chains. Moreover, HsaD from Mtb had 100-fold higher specificity (kcat/KM) for a metabolite with a partially-degraded side chain. Chapter 3 presents a mechanism for KstR2, a TetR family transcriptional repressor that regulates the HIP catabolic genes, including ipdABCF and echA20. The KstR2 dimer bound two equivalents of HIP-CoA with high affinity (KD = 80±10 nM). Crystallographic analyses revealed that HIP-CoA binding induces conformational changes in the dimer that preclude DNA-binding. Mutagenesis substantiated the roles of Arg162 and Trp166 in HIP-CoA binding. In Chapter 4, key HIP catabolic steps are elucidated. Two previously undescribed metabolites, 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH-HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) were identified using deletion mutants of ipdC and ipdAB, respectively, combined with novel metabolomics approaches. Together, purified IpdC, IpdF and EchA20 transformed 5-OH-HIC-CoA to COCHEA-CoA. These data, along with those from additional mutants, were used to formulate a HIP catabolic pathway and to predict that cholesterol catabolism yields four propionyl-CoA, four acetyl-CoA, one pyruvate, and one succinate. Chapter 5 establishes that IpdAB catalyzes a retro-Claisen-like ring-opening of COCHEA-CoA (kcat/KM = 2±0.7 × 10⁵ M⁻¹s⁻¹) despite structural similarity with Class I CoA transferases. Based on crystal structures of IpdAB and biochemical data, a mechanism for ring-cleavage is proposed in which conserved Glu105 acts as a catalytic base. Overall, this work significantly advances our understanding of bacterial steroid catabolism and facilitates the development of novel therapeutics to treat TB.

Text

2018-02-14

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64634

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/