Chimie et Biochimie des Substances Naturelles, UMR 5154 CNRS, Muséum National d'Histoire Naturelle, CP 54, 57 rue Cuvier, 75005 Paris, France.
Published: July 2007
AI Article Synopsis
Article Abstract
Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.
Microcin J25 (MccJ25), a lasso peptide antibiotic with a unique structure that resembles the lariat knot, has been a topic of intense interest since its discovery in 1992. The precursor (McjA) contains a leader and a core segment. McjB is a protease activated upon binding to the leader, and McjC converts the core segment into the mature MccJ25.
Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides.
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Sud, Université Paris-Saclay, 1 avenue de la Terrasse, 91190, Gif-sur-Yvette, France.
Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post-translational modification of the precursor McjA by the ATP-dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three-dimensional folding of MccJ25.
Molécules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS, Muséum National d'Histoire Naturelle, CP 54, 57 rue Cuvier, 75005 Paris, France.
Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight-residue macrolactam ring through which the C-terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC).
Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E.
