In the ongoing fight against antibiotic resistance, iron acquisition, and more specifically siderophore biosynthesis, has become an attractive target for discovery of new antibiotics.  Recent work with Mycobacterium tuberculosis has demonstrated the potential for small molecule inhibition of siderophore biosynthetic enzymes.  Siderophore biosynthesis in Bacillus anthracis has been shown to be important for spore outgrowth and pathogenicity. Therefore, targeted inhibition of the biosynthesis of the essential siderophore, petrobactin, may hold promise as a potential therapeutic against anthrax.  Our work has focused on characterizing the enzymes responsible for petrobactin biosynthesis and designing inhibitors for key biosynthetic steps.  In the process, we have discovered that the substrate specificity of these enzymes is highly flexible and can be exploited for specialty chemical production. 

Petrobactin

We performed a series of studies with the objective of dissecting the catalytic function of each of the six Asb enzymes.  Our initial studies focused on three enzymes, AsbC, AsbD, and AsbE, presumed to catalyze synthesis of the first intermediate in petrobactin biosynthesis (3, 4-dihydroxybenzoyl-spermidine). We have shown that AsbC is a 3, 4-dihydroxybenzoic acid-AMP ligase similar to EntE and VibE in enterobactin and vibriobactin biosynthesis.  AsbD was shown to be an acyl/peptidyl/aryl carrier protein similar to thiolation domains found in polyketide, non-ribosomal peptide, and siderophore biosynthetic complexes.  AsbE was shown to condense the AsbD-bound 3, 4-dihydroxybenzoic acid with spermidine, citryl-spermidine, and several polyamine analogs. Through substrate specificity assays, AsbC was shown to be capable of loading substrates with hydrogen bond donors at the meta and para positions of the benzyl ring onto holo-AsbD.  This finding was supported by our initial inhibition studies where nonydrolyzable acylsulfate analogs of DHB-AMP containing hydroxyl groups at these positions were capable of inhibiting ATP-³²PPi exchange and those without did not. 

Two condensation enzymes, AsbA and AsbB, have been shown to generate the amide bonds in the central portion of petrobactin.  AsbA has been shown by others to generate citryl-spermidine through an ATP-dependent condensation mechanism.  The order of the remaining condensations catalyzed by AsbB and AsbE is flexible, suggesting that these enzymes could be used to generate a variety of small molecules.  The last enzyme in the pathway, AsbF, is responsible for generating protocatechuate from an intermediate in the shikimate pathway. Assays are currently being developed for use in high throughput screens of chemical libraries to indentify inhibitors of each enzyme.  This presentation will demonstrate the novel petrobactin biosynthetic pathway, the flexibility of the participating enzymes, and progress towards identifying novel therapeutic leads against Bacillus anthracis.