Characterization and Analysis of Early Enzymes for Petrobactin Biosynthesis in Bacillus anthracis

Abstract

Recently, iron acquisition and, more specifically, enzymes involved in siderophore biosynthesis have become attractive targets for discovery of new antibiotics. Accordingly, targeted inhibition of the biosynthesis of petrobactin, a virulence-associated siderophore encoded by the asb locus in Bacillus anthracis, may hold promise as a potential therapy against anthrax. This study describes the biochemical characterization of AsbC, the first reported 3,4-dihydroxybenzoic acid-AMP ligase, and a key component in the biosynthesis of DHB-spermidine (DHB-SP), the first isolable intermediate in petrobactin biosynthesis. AsbC catalyzes adenylation to the corresponding AMP ester of the unusual precursor 3,4-dihydroxybenzoate, in addition to benzoate substrates bearing hydrogen bond-donating substituents at the para and meta positions on the phenyl ring. In a second reaction, AsbC catalyzes transfer of the activated starter unit to AsbD, an aryl carrier protein similar to acyl and peptidyl carrier proteins that function in fatty acid, polyketide, and nonribosomal peptide biosynthesis. A third protein, AsbE, is shown to be responsible for condensation of 3,4-dihydroxybenzoyl-AsbD with spermidine, providing the DHB-spermidine arms that are linked to citrate for assembly of petrobactin. On the basis of the selective substrate profile of AsbC, a nonhydrolyzable analogue of 3,4-DHB-AMP was synthesized and shown to effectively inhibit AsbC function in vitro.

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

“…Because abrogation of petrobactin production or uptake has been shown to severely attenuate B. anthracis virulence (6,10,15), enzymes in the biosynthetic pathway remain attractive targets for identification of new antimicrobials. Gaining deeper insights into the details of NIS-associated enzyme function might provide access to mechanism-based inhibitors, as have been demonstrated for nonribosomal peptide synthetase-derived siderophore adenylation domains (13,65,66). Moreover, development of the chemoenzymatic approach described in this work toward creating new siderophore analogs could be applied to generate a class of "Trojan horse" antibiotics, bearing improved target effects and IC 50 values compared with conventional chemotherapeutics (67,68).…”

“…1C) and its regioisomer N8- (3,4-dihydroxybenzoyl)-spermidine (compound 4Ј) (11,13) (Fig. 7, bottom trace).…”

“…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).…”

“…Once available, linkage of the 3,4-DHBA moieties to an exposed primary amine is facilitated by interactions of three other asb products; AsbC catalyzes adenylation of 3,4-DHBA followed by transfer to the phosphopantetheinylated aryl carrier protein AsbD. Subsequently, AsbE functions to catalyze amide bond formation between 3,4-DHBA and molecules bearing a primary amine (13) (Fig. 1C).…”

Phaeobacter gallaeciensis genomes from globally opposite locations reveal high similarity of adaptation to surface life

“…A Chrome Azurol S assay showed that both Phaeobacter strains excrete siderophores under iron-limiting conditions, whereas Roseobacter denitrificans, which lacks the gene cluster, shows no excretion (Figure 4). The organization of the gene cluster is identical to that of the asbABCDEF petrobactin biosynthesis locus of Bacillus anthracis and Marinobacter aquaeoeli VT8 (Pfleger et al, 2007;Homann et al, 2009). The only difference is that asbC and asbD are fused in P. gallaeciensis (Figure 4).…”