S1C), and addition of hydroxyl amine to the assay allowed trapping of C-terminally activated TglA as the hydroxamate ( fig. Performing the reaction in buffer made with H 2 18O and subsequent MS analysis demonstrated that the product contains one 18O atom ( fig. We next purified Cys-tRNA Cys and showed that TglB does not transfer the Cys to the C-terminus of TglA unless ATP is present, which is converted to ADP and phosphate ( fig. This C-terminal peptide extension not only constitutes a previously unknown posttranslational modification but also seems counterintuitive since a more logical route to the product appears to be encoding the Cys on tglA. coli confirming that TglB adds a Cys to the C-terminus of TglA in a tRNA dependent manner ( fig. 2A) as that isolated from co-expression in E. In vitro incubation with Cys, ATP, tRNA Cys and Cys tRNA synthetase (CysRS) resulted in the same product (TglA-Cys Fig. We expressed TglA and TglB individually as His 6-tagged proteins and purified them. High resolution tandem mass spectrometry analysis of the peptide suggested that the adduct was attached to the C-terminal alanine instead of the anticipated ester linkage to a Ser in the peptide ( Fig.
This increase is inconsistent with glutamylation, but could be the result of condensation with a cysteine residue.
Co-expression of His 6-TglA and TglB in Escherichia coli and subsequent purification of the peptide demonstrated an increase in mass by 103 Da ( Fig. maculicola ES4326, such a protein (TglB) is encoded near an open reading frame encoding a 50-amino acid peptide (TglA Fig. In the plant pathogen Pseudomonas syringae pv. ( D) Analysis of the TglB product by tandem electrospray ionization (ESI) mass spectrometry. (C) Matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectra of TglA coexpressed with TglB. Dha, dehydroalanine Dhb, dehydrobutyrine. Small LanB proteins lack the elimination domain. ( A) LanB enzymes glutamylate Ser/Thr residues and subsequently eliminate the glutamate to form dehydroamino acids. A survey of >100,000 publicly available bacterial genomes revealed more than 600 genes encoding LanB-like proteins in which the elimination domain is not present within the cluster or genome.įunction of a small LanB enzyme, TglB, found in P. Subsequently, the glutamate is eliminated to generate dehydroamino acids ( Fig. During their maturation, Ser and Thr residues are glutamylated by LanB enzymes in a glutamyl-tRNA dependent mechanism ( 5, 6). They are biosynthesized from a precursor peptide consisting of a leader peptide that serves as a recognition motif for the biosynthetic enzymes and a core peptide that is converted to the final product. Ribosomally-synthesized and posttranslationally-modified peptides (RiPPs) ( 3) include lantibiotics and thiopeptides that are used in food and agriculture ( 4).
In this study we focus on a group of biosynthetic gene clusters for which the final products were not known and could not be predicted. Historically these compounds have been discovered by activity-based screens, but an alternative avenue for their discovery starts with identification of their biosynthetic gene clusters now that bacterial genomes have revealed the tremendous diversity of natural products that remain to be discovered ( 2). Bacteria produce many small-molecule natural products that play important roles in communication, symbiosis, and competition ( 1).
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