The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) proceeds via the multistep maturation of genetically encoded precursor peptides, often catalyzed by enzymes with multiple functions and iterative activities. Recent studies have suggested that, among other factors, conformational sampling of enzyme:peptide complexes likely plays a critical role in defining the kinetics and, ultimately, the set of post-translational modifications in these systems. However, detailed characterizations of these putative conformational sampling mechanisms have not yet been possible on many RiPP biosynthetic systems. In this study, we report the first comprehensive application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study the biophysical properties of a RiPP biosynthetic enzyme. Using the well-characterized class II lanthipeptide synthetase HalM2 as a model system, we have employed HDX-MS to demonstrate that HalM2 is indeed a highly structurally dynamic enzyme. Using this HDX-MS approach, we have identified novel precursor peptide binding elements, have uncovered long-range structural communication across the enzyme that is triggered by ligand binding and ATP hydrolysis, and have detected specific interactions between the HalM2 synthetase and the leader- and core-peptide subdomains of the modular HalA2 precursor peptide substrate. The functional relevance of the dynamic HalM2 elements discovered in this study are validated with biochemical assays and kinetic analysis of a panel of HDX-MS guided variant enzymes. Overall, the data have provided a wealth of fundamentally new information on LanM systems that will inform the rational manipulation and engineering of these impressive multifunctional catalysts. Moreover, this work highlights the broad utility of the HDX-MS platform for revealing important biophysical properties and enzyme structural dynamics that likely play a widespread role in RiPP biosynthesis.