Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF-a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.AsbF structure ͉ dehydratase ͉ siderophore ͉ virulence factor S iderophore production in pathogenic bacteria has gained considerable attention because of its crucial function in essential iron uptake by many microbes and the relevance of siderophore-associated proteins as molecular markers of various infectious agents (1). In Bacillus anthracis, the causative agent of anthrax, 2 siderophores, petrobactin and bacillibactin (Fig. 1A), play a significant role during iron acquisition (2-4), but only petrobactin is absolutely essential for full virulence within a mammalian host (5). This siderophore was initially discovered from the Gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, whose genome bears a biosynthetic gene cluster homologous to the B. anthracis asb operon (2). Recent genetic and chemical analysis suggests that petrobactin biosynthesis may also be a prerequisite for virulence in related Bacillus species (6). These studies highlight the importance of elucidating the mechanisms of siderophore production in pathogenic microbes as a target for abrogating infection by organisms like B. anthracis, a rapidly virulent microbe with proven potential as a bioterrorism agent. Based on these factors, we have initiated studies to investigate key biosynthetic enzymes for petrobactin assembly in efforts to establish new antimicrobial targets t...

Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 µM and 19 µM against SbnE and 180 µM and 200 µM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus, B. anthracis, E. coli and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents

Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results: Purified AsbA-E proteins reconstituted petrobactin assembly in vitro. The crystal structure and enzymatic studies of AsbB highlight its function and role in the siderophore pathway. Conclusion: AsbB characterization demonstrated reaction flexibility and substrate positions in the binding pocket. Significance: Siderophore synthetases represent promising antimicrobial targets, and characterization of these versatile enzymes enables creation of novel compounds.

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