title: The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.
abstract: The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.
authors: Department of Fundamental Microbiology, University of Lausanne, Lausanne Switzerland.
authors: Nelson
authors: N
authors: Rojas Murcia
authors: Department of Fundamental Microbiology, University of Lausanne, Lausanne Switzerland.
authors: Xiaoyun
authors: X
authors: Lee
authors: Protein Analysis Facility, University of Lausanne, Lausanne Switzerland.
authors: Patrice
authors: P
authors: Waridel
authors: Department of Fundamental Microbiology, University of Lausanne, Lausanne Switzerland.
authors: Alessandro
authors: A
authors: Maspoli
authors: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA USA.
authors: Heidi J
authors: HJ
authors: Imker
authors: Department of Fundamental Microbiology, University of Lausanne, Lausanne Switzerland.
authors: Tiancong
authors: T
authors: Chai
authors: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA USA.
authors: Christopher T
authors: CT
authors: Walsh
authors: Department of Fundamental Microbiology, University of Lausanne, Lausanne Switzerland.
authors: Cornelia
authors: C
authors: Reimmann
keywords: Pseudomonas; oxyvinylglycine; secondary metabolite; thiotemplate; toxin