Detailed Analysis of Biosynthetic Components for the Virulence-Associated Siderophore Petrobactin from Bacillus anthracis.

Abstract

Iron is an essential cofactor in biology, yet most organisms’ acquisition of iron is hampered by inaccessibility of this metal in ferric complexes. Thus, life on Earth has developed diverse strategies to obtain necessary levels of free iron, one of the most prominent among microbes being the biosynthesis of specific, high-affinity chelators called siderophores. Bacillus anthracis, the causative agent of anthrax, requires the siderophore petrobactin for full virulence. Prior studies have demonstrated the asbABCDEF operon encodes biosynthetic machinery for this secondary metabolite: The virulence-associated “NRPS-independent siderophore (NIS) synthetase” protein family includes the enzymes AsbA and AsbB, which condense the common metabolites citrate and spermidine; meanwhile, the AsbCDE complex promiscuously transfers 3,4-dihydroxybenzoic acid (3,4-DHBA) to primary amines. 3,4-DHBA moieties of petrobactin allow the siderophore to evade neutralization by innate immune mechanisms, yet the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the asb operon, asbF, has remained unclear. The data presented herein reveals that the primary metabolite 3-dehydroshikimate is converted to 3,4-DHBA via AsbF catalysis. Subsequent mass spectrometric studies demonstrate that five gene products encoded by the asb operon are necessary and sufficient for conversion of endogenous metabolic precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. Structural characteristics of AsbB were applied to provide new insight into how this enzyme, and its partner synthetase AsbA, can bind and adenylate multiple citrate-containing substrates, followed by incorporation of both natural and unnatural polyamine nucleophiles. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides indicate two products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. Combined, these studies establish new avenues for the chemoenzymatic synthesis of novel compounds and investigate key biosynthetic enzymes of petrobactin assembly with the purpose of promoting better understanding of bacterial host iron acquisition and identifying new antimicrobial strategies to protect against B. anthracis and other pathogenic bacteria.