Natural products (NPs) account for a large number of human pharmaceuticals, yet many of these compounds are structurally complex and difficult to chemically synthesize with the desired purity and scale. For this reason, a thorough understanding of the enzymatic mechanisms of NP biosynthesis are needed to unlock more sustainable routes for the synthesis and structural engineering of NPs of biomedical relevance. My work has focused on understanding the biosynthesis of the antimicrobial lanthipeptides, which are constructed in multistep reactions by enzymes (lanthipeptide synthetases) with relaxed substrate specificity. The reaction catalyzed by class II lanthipeptide synthetase (LanM) enzymes involves dehydration of serine/threonine residues in the LanA precursor peptide, followed by intramolecular addition of cysteine thiols to form thioether bridges. LanM enzymes function in an iterative manner, whereby multiple thioether linkages are installed in a sequential fashion into the maturing LanA intermediate. Collectively, LanM enzymes differ dramatically in their substrate specificities, catalytic efficiencies, and other biochemical properties. The mechanisms underlying this functional heterogeneity remain enigmatic. This talk highlights recent efforts by our lab to untangle the complex relationships between structural dynamics, conformational changes, and biochemical function in LanM enzymes using mass spectrometry-based approaches. Using the haloduracin synthetase (HalM2) as a model LanM system, mass spectrometry-based kinetic studies show that conformational changes and enzyme:peptide interactions influence the kinetics and sequence of biosynthetic events. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies show that HalM2 is a dynamic enzyme that undergoes local structural rearrangements and long-range allosteric signalling upon peptide or nucleotide binding. Mutations to previously overlooked, dynamic HalM2 structural elements impact the catalytic activities (dehydration and cyclization) of HalM2, providing evidence that conformational changes and enzyme-peptide interactions contribute to function. Finally, native mass spectrometry coupled to ion mobility shows that HalM2 exists in multiple three-dimensional conformations, and that this conformational landscape is influenced by HalA2 precursor peptide binding and enzyme mutagenesis. Cumulatively, our studies illustrate the many benefits provided by mass spectrometry for characterizing the mechanistic and structural properties of RiPP biosynthetic enzymes.