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Abstract Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbialVibriospecies. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of fourl-Ser residues, of which onel-Ser is appended by a fatty acid and the remainingl-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant ofVibrio campbelliispecies. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin. Graphical abstract
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In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii
Siderophores produced via non-ribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. An improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central L-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of L-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of L-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of L-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of L-Ser, DHB, and L-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (L-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS ‘branching’ in the presence of a terminating C-T-C motif (FbsG).
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- Award ID(s):
- 1654611
- PAR ID:
- 10357526
- Editor(s):
- He, Chuan; Seyedsayamdost, Mohammed R.
- Date Published:
- Journal Name:
- ACS Chemical Biology
- ISSN:
- 1554-8929
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acschembio.2c00573
