title: 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.affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Tyler D
authors.initials: TD
authors.lastname: Nusca
authors.forename: Youngchang
authors.initials: Y
authors.lastname: Kim
authors.forename: Natalia
authors.initials: N
authors.lastname: Maltseva
authors.forename: Jung Yeop
authors.initials: JY
authors.lastname: Lee
authors.forename: William
authors.initials: W
authors.lastname: Eschenfeldt
authors.forename: Lucy
authors.initials: L
authors.lastname: Stols
authors.forename: Michael M
authors.initials: MM
authors.lastname: Schofield
authors.forename: Jamie B
authors.initials: JB
authors.lastname: Scaglione
authors.forename: Shandee D
authors.initials: SD
authors.lastname: Dixon
authors.forename: Daniel
authors.initials: D
authors.lastname: Oves-Costales
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Challis
authors.forename: Philip C
authors.initials: PC
authors.lastname: Hanna
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011073:Polyamines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D017262:Siderophores / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001549:Benzamides; D011073:Polyamines; D017262:Siderophores; C450699:petrobactin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases