Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition

Abstract

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death. Synthesis of lipids critical for Mtb’s cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4’-phosphopantetheine (Ppt) from coenzyme A to diverse acyl carrier proteins (ACPs). We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss of function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor co-crystal structure may aid further development of anti-mycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis

“…Soluble, active protein was overexpressed by removing 20 amino acids from the C‐terminus while changing from BL‐21 DE3 to the BL‐21 AI strain further improved yields and mitigated the toxicity associated with the protein's expression without induction. This purified protein was used in the biochemical analysis that confirmed its activity as a Ppt hydrolase …”

“…Of the 12 colonies we identified that were resistant to the lead molecule 8918, 10 contained mutations within the rv2795c ( pptH ) gene. The fact that the same resistance phenotype was observed in the pptH deletion strains suggested that these mutations were LOF, and the presence of a WT pptH copy restored the sensitivity of resistant mutant Mtb cells to 8918 . However, without structural knowledge, the specific impact of these mutations on PptH was speculative.…”

“…The 2.5 Å structure revealed that the enzyme is a member of the MPE superfamily, featuring the characteristic binuclear center within the active site while the peripheral loops and helices are distinct from those of previously characterized MPEs. These findings have laid the groundwork for proposals on the general catalytic mechanism and the rationalization of the previously reported PptH mutations associated with 8918‐resistance while allowing for more informed comparisons between the PptH and AcpH‐type PpT hydrolases.…”

“…Leblanc et al established that PptT is essential during both the acute and chronic phases of infection in a mouse model . Ballinger et al reported the druggability of PptT by the amidinourea 8918, further designating it as a promising drug target …”

“…All species within the Mycobacterium genus possess a PptH ortholog, suggesting that this activity grants a fitness advantage to mycobacteria. Proposals of its physiological roles include the elimination of specific holo‐carrier proteins, which may be important during different metabolic stages, as well as a potential role in the recycling of pantothenate . However, much like the aforementioned AcpHs, the exact physiological role and regulation of PptH are not fully understood.…”