Functional and structural analysis of the siderophore synthetase AsbB through reconstitution of the petrobactin biosynthetic pathway from Bacillus anthracis.

Abstract

Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracis siderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds.

• Authors: Tyler D Nusca, Youngchang Kim, Natalia Maltseva, Jung Yeop Lee, William Eschenfeldt, Lucy Stols, Michael M Schofield, Jamie B Scaglione, Shandee D Dixon, Daniel Oves-Costales, Gregory L Challis, Philip C Hanna, Brian F Pfleger, Andrzej Joachimiak, David H Sherman
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 287 Issue 19 Pg. 16058-72 (May 04 2012) ISSN: 1083-351X [Electronic] United States
• PMID: 22408253 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Bacterial Proteins
• Benzamides
• Polyamines
• Siderophores
• petrobactin
• Ligases
• Carbon-Nitrogen Ligases

Topics

• Amino Acid Sequence
• Bacillus anthracis (genetics, metabolism)
• Bacterial Proteins (chemistry, genetics, metabolism)
• Benzamides (chemistry, metabolism)
• Biocatalysis
• Biosynthetic Pathways
• Carbon-Nitrogen Ligases (chemistry, genetics, metabolism)
• Crystallography, X-Ray
• Ligases (chemistry, genetics, metabolism)
• Models, Chemical
• Models, Molecular
• Molecular Sequence Data
• Molecular Structure
• Polyamines (chemistry, metabolism)
• Protein Binding
• Protein Structure, Tertiary
• Sequence Homology, Amino Acid
• Siderophores (chemistry, metabolism)
• Substrate Specificity