Structural and functional analysis of AsbF: Origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis

Abstract

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...

Characterization of a Bacillus subtilis transporter for petrobactin, an anthrax stealth siderophore

“…The structure was determined by SAD phasing using HKL3000 as described previously (48). The initial model was built using the arp/Warp, routine of HKL3000 and Coot, and the subsequent refinement was performed iteratively by phenix.refine and Coot until it converged to the R factor of 0.158, and the free R of 0.199.…”

Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis

“…Most other catecholate siderophores contain 2,3-dihydroxybenzoic subunits, which are effectively neutralized by this protein during the host response to infection (27)(28)(29). Previous work has shown that asbF, the final gene of the petrobactin biosynthetic operon, encodes a dehydratase responsible for conversion of 3-dehydroshikimate to 3,4-DHBA (10,14) (Fig. 1C).…”

“…The broad eubacterial distribution of NIS systems (10,23,25) indicate that new information about petrobactin biosynthesis and transport will provide insights into these iron acquisition strategies. In this work, we sought to resolve remaining questions about the petrobactin biosynthetic pathway.…”

“…Conversely, a second siderophore, petrobactin (Fig. 1A), is essential for virulence within current infection models (6,10) and has since become a focus of multiple microbiological, genetic, and biochemical studies (3,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)20). This mixed catechol-carboxylate siderophore was first isolated from the Gram-negative marine microbe Marinobacter hydrocarbonoclasticus (21).…”

Genetic analysis of petrobactin transport inBacillus anthracis

“…Because it is known that mutants unable to synthesize petrobactin exhibit significant attenuation in murine virulence (Cendrowski et al, 2004;Pfleger et al, 2008), we A. Extracellular catechol levels were measured over time in culture supernatants using the Arnow assay. Data were normalized to the OD600 of cultures at each time point and are presented as per cent of wild-type petrobactin levels at 3 h. No signal could be detected at time points prior to 3 h. Error bars represent standard deviation of triplicate samples within one experiment.…”