Dissecting Reactions of Nonlinear Precursor Peptide Processing of the Class III Lanthipeptide Curvopeptin

Abstract

Lanthipeptides are ribosomally synthesized peptides which undergo extensive post-translational modifications. In addition to novel structural features and bioactivities, the in vitro study on the biosynthesis of the class III lanthipeptide labyrinthopeptin revealed a unique C- to N-terminal directionality of biosynthetic processing. The recently described class III lanthipeptide curvopeptin allowed investigating the directionality aspect in much greater detail: Structural characterization of nine curvopeptin biosynthesis intermediates by high-resolution mass spectrometry combined with a deuterium-labeling approach enabled for the first time building a comprehensive biosynthesis model featuring all three post-translational modification reactions: phosphorylation, elimination, and cyclization. These results point to a nonlinear processing scheme with a predominant C → N-terminal directionality. Our data give important mechanistic insights into the concerted processing and directionality of the multifunctional class III modifying enzymes. The data are of significance in the light of obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of ribosomally synthesized and post-translationally modified peptides.

Structure and tRNA Specificity of MibB, a Lantibiotic Dehydratase from Actinobacteria Involved in NAI-107 Biosynthesis

“…Our data then implies Ser5 to be dehydrated last. Based on the collected information MibB dehydrates the precursor peptide MibA without strict directionality (Figure S2C), an observation that has also been reported for other lanthipeptide biosynthetic systems (Jungmann et al, 2014, Mukherjee and van der Donk, 2014). …”

“…NisB dehydrates NisA in a mostly N-to-C terminal fashion (Lubelski et al, 2009, Zhang et al, 2014), whereas MibB processed MibA following a mostly C-to-N terminal directionality after the first dehydration occurred at the N-terminus. Similar order of post-translational modifications that are not strictly directional has been reported for other RiPP biosynthetic enzymes (Jungmann et al, 2014, Melby et al, 2012). Interestingly, during the initial steps of microbisporicin biosynthesis the C-terminus of MibA must undergo oxidative decarboxylation presumably catalyzed by the enzyme MibD (Foulston and Bibb, 2010).…”

Product Formation by the Promiscuous Lanthipeptide Synthetase ProcM is under Kinetic Control

“…10,26–28 Thus, a possible explanation for the unusual selectivity of the ring formation in the structurally diverse prochlorosins is that the ring patterns are determined by the order of dehydration and cyclization. The order of dehydration has been established in a previous study for ProcA2.8 and ProcA3.3.…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…A third route to (Me)Lan formation in class III and IV lanthipeptides features the use of trifunctional enzymes composed of a central Ser/Thr kinase domain, an N-terminal phosphoSer/phosphoThr lyase domain, and a C-terminal cyclization domain (Goto et al, 2010, Iftime et al, 2015, Jungmann et al, 2014, Müller et al, 2010). Hence, these enzymes use the same chemical strategy as the class II synthetases, but like the class I dehydratases, activation of the Ser/Thr hydroxyl groups and elimination of the activating group is achieved in separate active sites.…”