title: Dissecting the maturation steps of the lasso peptide microcin J25 in vitro.
abstract: 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). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP-dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched-cyclic topology could be generated in vitro.
authors.affiliation: Molécules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS, Muséum National d'Histoire Naturelle, CP 54, 57 rue Cuvier, 75005 Paris, France.
authors.forename: Kok-Phen
authors.initials: KP
authors.lastname: Yan
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Li
authors.forename: Séverine
authors.initials: S
authors.lastname: Zirah
authors.forename: Christophe
authors.initials: C
authors.lastname: Goulard
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Knappe
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rebuffat
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001430:Bacteriocins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; C020411:microcin