Enzymatic logic of anthrax stealth siderophore biosynthesis: AsbA catalyzes ATP-dependent condensation of citric acid and spermidine

Petrobactin is an iron-chelating siderophore originally isolated from Marinobacter hydrocarbonoclasticus that has been shown to play an important role in growth under iron-deficient conditions and virulence of the deadly bioterrorism agent Bacillus anthracis. It has recently been shown not to bind to siderocalin, leading it to be designated as a "stealth siderophore" that can avoid the mammalian immune system. A unique combination of nonribosomal peptide synthetase (NRPS) and NRPS-independent siderophore (NIS) synthetase enzymes is known to be required for petrobactin biosynthesis in B. anthracis. Here it is shown that AsbA from B. anthracis, the first type A NIS synthetase to be biochemically characterized, catalyzes ATP-dependent regioselective condensation of citric acid with N⁸ of spermidine, but not with N¹-(3,4-dihydroxybenzoyl)-spermidine. These results rule out a recently proposed pathway for petrobactin biosynthesis involving AsbA-catalyzed condensation of N¹-(3,4-dihydroxybenzoyl)-spermidine with citric acid and show that acylation of N¹ of spermidine with the 3,4-dihydroxybenzoyl group must occur after acylation of N⁸ of spermidine with citrate. They also provide the fundamental knowledge needed to establish a high throughput screen for inhibitors of AsbA that may provide the basis for development of new antibiotics for the treatment of deadly anthrax infections.