A D-Phenylalanine-Benzoxazole Derivative Reveals the Role of the Essential Enzyme Rv3603c in the Pantothenate Biosynthetic Pathway of Mycobacterium tuberculosis

New drugs and new targets are urgently needed to treat tuberculosis. We discovered the D-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis ( Mtb ) in multiple media and in macrophage infections. Metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/CoA 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 Mtb . The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum , but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an ortholog of PanG KPR, and demonstrated that purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112 -treated bacteria, indicating that Q112 has an additional target(s). Q112 -resistant strains contain loss-of-function mutations in the twin arginine translocaseTatABC, further underscoring Q112 s unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.Graphical AbstractNew antibiotics are urgently needed to treat tuberculosis. Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) . We find that Q112 inhibits Rv3603c, an essential enzyme of previously unknown function now identified as PanG. This enzyme catalyzes conversion of ketopantoate to pantoate, a key step in the biosynthesis of CoA. However, PanG inhibition cannot account for antibacterial activity and additional Q112 targets remain to be identified.