Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

N‐substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well‐studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide‐induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA‐ and acyl carrier protein‐dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanKI, PanKII and PanKIII) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanKI enzymes accept them as substrates, the highly selective PanKIIIs are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanKII) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.