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Open Access Highly Accessed Research article

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

Christina Ramel1, Micha Tobler1, Martin Meyer1, Laurent Bigler2, Marc-Olivier Ebert3, Barbara Schellenberg1 and Robert Dudler1*

Author Affiliations

1 Institute of Plant Biology and Zurich-Basel Plant Science Center, University of Zurich, Zurich, Switzerland

2 Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland

3 Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland

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BMC Biochemistry 2009, 10:26  doi:10.1186/1471-2091-10-26

Published: 28 October 2009

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 NaH13CO3 revealed preferential 13C-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.