Lantibiotics are ribosomally-synthesized, post-translationally modified peptide natural products that often exhibit antimicrobial activity. Lantibiotics typically function by inhibiting several highly conserved steps in bacterial peptidoglycan biosynthesis and/or by disrupting cell wall and plasma membrane integrity. Due to the fact that they target such essential components of bacterial cell viability, lantibiotics hae avoided common modes of bacterial resistance and have proven to be useful antibiotics against several dangerous Gram- positive human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci. Recognizing the potential of lantibiotics as alternative treatments for drug resistant bacterial pathogens, a renewed interest in the discovery and biosynthesis of these peptide natural products has blossomed over the past decade. This work has led to the discovery of many lantibiotic biosynthetic gene clusters and to the biochemical characterization of several of the biosynthetic enzymes. The post-translational modifications (PTMs) that are common to all lantipeptides include the ATP-dependent dehydration of serine and threonine residues in the peptide substrate (LanA) to form dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, respectively, followed by intramolecular attack of cysteine residues onto the Dha/Dhb residues to form lanthionine (Lan) and methyl lanthionine (MeLan) thioether rings. Usually, multiple Lan/MeLan rings are present in the final product and remarkably, for some lantibiotics, a single biosynthetic enzyme (LanM) installs all of these PTMs. Due to the inherent kinetic complexity in these systems, the molecular mechanisms underlying the timing of the various PTMs and the selection of specific dehydration and cyclization patterns by LanM are largely unknown. This information is expected to be critical for successful manipulation of these enzymes as catalysts to engineer novel lantipeptide structures in drug development efforts. The long term goal of this proposal is to elucidate the kinetic basis for LanM catalysis and to identify kinetic and structura features of the system that govern substrate specificity. To achieve this goal, several innovative single molecule fluorescence assays will be developed to kinetically characterize LanM/LanA systems in unprecedented detail. These assays will provide kinetic information for individual sub-steps in the multi-step LanM-catalyzed reaction, reveal the presence (or absence) of heterogeneity in the pattern of post-translational modification events, and define the role of dynamic protein-protein interactions between LanM and LanA in determining the preferred PTM reaction pathway. The assay systems and methods of analysis to be developed herein can be applied to many LanM/LanA systems to uncover the general and unique catalytic features of each system, as well as towards kinetic studies of other peptide-modifying natural product biosynthetic systems. PUBLIC HEALTH RELEVANCE: New antibiotic compounds are needed to mitigate the daunting treat to human health posed by drug resistant bacterial pathogens. Lantibiotics are a promising class of peptide natural products that avoid common bacterial resistance mechanisms. This proposal seeks to gain a detailed mechanistic understanding of the enzymes that construct lantibiotics, in hopes of better manipulating their activity for the engineering or designed synthesis of novel compounds with new or improved biological activity.