The first focus is on enterobacterial strains that turn on biosynthetic genes for the siderophore enterobactin in microenvironments where iron is limiting, including vertebrate hosts. Enterobactin binds ferric iron avidly and is subjected to specific uptake by the producing bacteria. Some pathogenic gram negatives further elaborate the enterobactin scaffold to make C-glucosylated forms, also known as salmochelins from their discovery in iron-scavenging salmonella typhi strains. Salmochelin-producing bacteria tailor the Ent scaffold under direction of the Iro gene cluster IroBCDEN. We will study the enzymatic mechanism for C- glucosylation by IroB and determine to what extent the glucosylation of the dihydroxybenzene rings of the Ent scaffold interfere with sequestration of the siderophore by the mammalian host protein siderocalin. Failure to sequester the modified enterobactins should correlate with increased pathogenicity of the bacteria. The second focus is on the productuiion of the siderophore acinteobactin by the gram negative respiratory pathogen Acinetobacter baumanii. Siderophore production correlates with increased virulence. Acinetobactin has all three known-iron chelating groups, catechol, thiazoline, and hydroxamate, built into its skeleon by a nonribosomal peptide synthetase assembly line. The six genes BasABCDEF encode the siderophore synthetase assembly line. We plan to overproduce each protein and evaluate the following unusual featiures predicted for assembly line ooperations: action of two free standing adenylation and thiiolation domains, cyclodehdration by tandem condensation domains, hydroxamate formation during chain termination in siderophore maturation. Characterization of the salrnochelin and acinetobactin biosynthesizing enzymes will be the foundation for subsequent evaluation of enzyme inhibitors that might block siderophore production.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Macromolecular Structure and Function A Study Section (MSFA)
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Korpela, Jukka K
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Harvard University
Schools of Medicine
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Wencewicz, Timothy A; Walsh, Christopher T (2012) Pseudomonas syringae self-protection from tabtoxinine-?-lactam by ligase TblF and acetylase Ttr. Biochemistry 51:7712-25
Parker, Jared B; Walsh, Christopher T (2012) Stereochemical outcome at four stereogenic centers during conversion of prephenate to tetrahydrotyrosine by BacABGF in the bacilysin pathway. Biochemistry 51:5622-32
Parker, Jared B; Walsh, Christopher T (2012) Olefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis. Biochemistry 51:3241-51
Walsh, Christopher T; Fischbach, Michael A (2010) Natural products version 2.0: connecting genes to molecules. J Am Chem Soc 132:2469-93
Fischbach, Michael A; Walsh, Christopher T (2009) Antibiotics for emerging pathogens. Science 325:1089-93
Heemstra Jr, John R; Walsh, Christopher T; Sattely, Elizabeth S (2009) Enzymatic tailoring of ornithine in the biosynthesis of the Rhizobium cyclic trihydroxamate siderophore vicibactin. J Am Chem Soc 131:15317-29
Wuest, William M; Sattely, Elizabeth S; Walsh, Christopher T (2009) Three siderophores from one bacterial enzymatic assembly line. J Am Chem Soc 131:5056-7
Koglin, Alexander; Lohr, Frank; Bernhard, Frank et al. (2008) Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454:907-11
Calderone, Christopher T; Iwig, David F; Dorrestein, Pieter C et al. (2007) Incorporation of nonmethyl branches by isoprenoid-like logic: multiple beta-alkylation events in the biosynthesis of myxovirescin A1. Chem Biol 14:835-46
Zhou, Zhe; Lai, Jonathan R; Walsh, Christopher T (2007) Directed evolution of aryl carrier proteins in the enterobactin synthetase. Proc Natl Acad Sci U S A 104:11621-6

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