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Results and DiscussionSite-directed mutagenesis was performed on the pTUC202 plasmid (containing the MccJ25biosynthesis cluster) by using the QuickChange XL Site-Directed Mutagenesis Kit (AgilentTechnologies). The plasmid carrying mutations was transformed into a producing strain,which was then cultured in M63 medium to assess the production of mature MccJ25 in theculture supernatant by LC-MS detection.Sequence analysis of McjB enabled to hypothesize that McjB could be a proteaseresponsible for the cleavage of the precursor McjA [2]. Site-directed mutagenesis allowedto show that McjB is actually a cysteine protease, where Cys150, His182 and Glu186 arecrucial residues for its activity, probably constituting a catalytic triad. The involvement ofCys150 of McjB in the maturation process has been confirmed in vitro by analyzing theproduction of MccJ25 from recombinant McjA in the presence of either recombinant McjBor McjB[C150A], and recombinant McjC, ATP and Mg 2+ .The homologue of McjC, the asparagine synthetase, catalyses the activation of thecarboxyl group via an acyl-AMP intermediate and subsequent formation of the amide bond.Sequence analysis of McjC revealed conserved residues involved in the binding of ATPand Mg 2+ [2]. Site-directed mutagenesis of these residues (Ser199, Asp203 and Asp302)confirmed that they are crucial for McjC activity. It thus provides evidence that McjC isresponsible for the activation of the side chain of Glu8 of MccJ25, which is a pre-requiredstep for the macrolactam ring formation.Therefore, the study shows that the biosynthesis of MccJ25 requires the involvementof two enzymes: one is a cysteine protease (McjB), which cleaves the precursor McjA,while the other (McjC) activates the substrate possibly by formation of an acyl-AMPintermediate. Prior to the macrolactam ring closure, the folding of the precursor must occurso that the C-terminal tail can be properly trapped within it (Figure 1B). However the orderof these different steps remains to be determined.Our work provides insights into the biosynthesis of lasso peptides. Such knowledgewould probably have important biotechnological impacts, as the lasso structure incombination with its maturation enzymes could be used to generate peptides endowed withnovel bioactivities. The understanding of the mechanisms underlying the formation of thelasso structure is therefore a major step towards the engineering of lasso peptides.AcknowledgmentsThis work was supported by the ANR project n BLAN-NT09-692063. We acknowledge the massspectrometry platform at the MNHN for access to the ESI-Qq-TOF spectrometer as well as thebacteriology facility.References1. Rebuffat, S., Blond, A., Destoumieux-Garzón, D., Goulard, C., Peduzzi, J. Curr. Protein Pept. Sci.5, 383-391 (2004).2. Duquesne, S., Destoumieux-Garzón, D., Zirah, S., Goulard, C., Peduzzi, J., Rebuffat, S. Chem. Biol.14, 793-803 (2007).3. Knappe, T.A., Linne, U., Zirah, S., Rebuffat, S., Xie, X., Marahiel, M.A. J. Am. Chem. Soc. 130,11446-11454 (2008).4. Salomón, R.A., Farías, R.N. J. Bacteriol. 174, 7428-7435 (1992).5. Rosengren, K.J., Clark, R.J., Daly, N.L., Goransson, U., Jones, A., Craik, D.J. J. Am. Chem. Soc.125, 12464-12474 (2003).397