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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Towards Lasso Peptide Engineering: Insights into theMaturation Mechanism of Microcin J25Kok-Phen Yan, Séverine Zirah, Yanyan Li, Christophe Goulard, andSylvie RebuffatMuséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique,Laboratoire Molécules de Communication et Adaptation des Microorganismes,FRE 3206 CNRS-MNHN, 75005, Paris, FranceIntroductionLasso peptides are knotted structures of bacterial origin, consisting of 16-21 residues,where an N-terminal macrolactam ring (resulting from the condensation of the N-terminalamine with a side chain carboxylate at position 8-9) traps the C-terminal tail of the peptidewithin it [1]. These peptides are remarkably resistant to proteases and denaturing agentsand show diverse biological activities such as receptor antagonists or enzyme inhibitors,resulting for some of them in antimicrobial activities. Such properties thus confer lassopeptides a high biotechnological interest. It is therefore essential to understand themechanisms of their biosynthesis. Lasso peptides are biosynthesized from a gene-encodedprecursor that is processed by two maturation enzymes [2,3]. In this study, we usedmicrocin J25 (MccJ25) [4,5] as a model peptide to unveil the mechanisms underlying thebiosynthesis of lasso peptides. MccJ25 is synthesized as a linear 58 amino acid precursor(McjA) containing a leader region in its N-terminal part, which is processed by twomaturation enzymes (McjB and McjC) (Figure 1A). In a previous study, we were able tosynthesize mature MccJ25 in vitro upon incubation of McjA with McjB and McjC in thepresence of ATP and Mg 2+ [2]. McjB shows weak similarities to putative transglutaminasesthat belong to the cysteine protease family. McjC is homologous to proteins involved in theformation of amide bonds (asparagine synthetases and β-lactam synthetases). We thereforehypothesized that McjB could be responsible for the cleavage of the precursor McjA andthat McjC could be involved in the formation of the macrolactam ring. In order to examinethis hypothesis, we used a site-directed mutagenesis approach in order to study the role ofthe McjA leader peptide and to confirm the respective roles of McjB and McjC.Fig. 1. A. Genetic system encoding MccJ25; B. Schematic view of MccJ25 maturation (theorder of the 3 steps in brackets remains to be determined).396