A d-Phenylalanine-Benzoxazole Derivative Reveals the Role of the Essential Enzyme Rv3603c in the Pantothenate Biosynthetic Pathway of .

New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole displays potent antibacterial activity against () in multiple media and in macrophage infections. A metabolomic profiling indicates that has a unique mechanism of action. perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in . The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close relative , but IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue -treated bacteria, indicating that has an additional target(s). -resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring 's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that resistance may derive from a decrease in uptake.