Dataset Information

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

ABSTRACT: BACKGROUND: Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for. RESULTS: We have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH(13)CO(3) revealed preferential (13)C-labeling at the ureido carbonyl position. CONCLUSION: The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

SUBMITTER: Ramel C

PROVIDER: S-EPMC2773804 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

Ramel Christina C Tobler Micha M Meyer Martin M Bigler Laurent L Ebert Marc-Olivier MO Schellenberg Barbara B Dudler Robert R

BMC biochemistry 20091028

<h4>Background</h4>Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a sec ...[more]

PMID: 19863801

Similar Datasets

Project description:As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.

Dataset Information

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

Publications

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets