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.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen often affecting patients who suffer from compromised antimicrobial barriers, as for instance the genetic disease cystic fibrosis (Lyczak et al, 2002)
Using in vitro methods and analysis of enzyme-attached substrates and pathway intermediates by mass spectrometry (MS), we identified the building blocks of AMB biosynthesis and we present a model of how the AMB precursor tripeptide may be assembled
non-ribosomal peptide synthetases (NRPSs) can be dissected into modules, each module being responsible for the incorporation of one amino acid or carboxy acid into the growing peptide chain
Summary
Pseudomonas aeruginosa is an opportunistic pathogen often affecting patients who suffer from compromised antimicrobial barriers, as for instance the genetic disease cystic fibrosis (Lyczak et al, 2002). One of the many toxins that this bacterium produces is L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), a non-proteinogenic amino acid belonging to a small group of natural compounds known as oxyvinylglycines (Berkowitz et al, 2006). Other members of this group are aminoethoxyvinylglycine (AVG), dimethyliminooxyvinylglycine, and guanidinooxyvinylglycine from Streptomyces spp. Oxyvinylglycines irreversibly inhibit pyridoxal phosphate (PLP)-dependent enzymes and have multiple targets in bacteria, animals, and plants (Berkowitz et al, 2006). A prominent plant target is the ethylene biosynthesis enzyme ACC synthase, which is inhibited by AVG. Available under the name of Retain®, AVG is widely used for the regulation of fruit set in orchard crops
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