Siderophore Synthetases in Three Bacterial Pathogens

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EXCEED THE SPACE PROVIDED. Iron chelators are synthesized, exported and retrieved by bacteria in iron-deficient microenvironments aid thus chelatars, also known as siderophores, serve as virulence factors for infections in vertebrates by pathogens such as Yersinia pestis in plague, Vibrio cholerae in cholera epidemics, and Pseudomonas aeruginosa in lung infection in CF patients. The respective siderophores, yersiniabactin (Ybt), vibriobactin(Vib), and pyochelin(Pch),are produced by non-ribosomal peptide synthetases(NRPS),activated by iron depletion. Yersiniabactin is assembled as a hybrid of a nonribosornal peptide and a polyketide (PK) by a mixed NRPS/PKS assembly line. The NRPS/PKS and PKS/NRPS switchpointsof this assembly line will be examined with purified multimodular subunits of Ybt synthetase. Ybt also has a tandem bithiazoline ring system and unusual C-methylations from Cyclization(Cy) and C-methylation(MT) domains embedded in the 17 domain Ybt assembly line. The mechanisms of the Cy and MT catalytic domains will be examined. The Pch assembly line involves four proteins and a cascade of acyl-S-enzyme intermediates that will be analyzed for timing and locus of specific chain elongation steps, including heterocyclic thiazoline ring reduction to thiazolidine and then subsequent N-methylationby the PchG subunit and the N-MT domain of the PchF subunit respectively. Because of the similarity of the Ybt and PCh siderophores protein-protein recognitionof domains and modules within and between the Pch and Ybt assembly lines will be examined to begin to establish rules for combinatorial biosynthesis of such NRP molecules. The third pathogenic siderophore system, the Vib synthetase assembles a branched siderophore and produces an oxazoline ring (from threonine) rather than the thiazoline riongs (from cysteine) in Pch and Ybt. There are two Cy domains and three Condensation (C) domains in the Vib assembly line and the function of all five of thesechain-elongation catalytic domains will be examined by biochemical analysis and mutagenesis to decipher the molecular logic of these NR.PS assemblies. Understanding of mechanism, e.g. of the heterocyclizing Cy domains may allow inhibitor design to block siderophore production by these multimodular enzymes.