The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multiple chemical processes are poorly understood. The lacticin 481 synthetase (LctM) cleaves eight chemical bonds and forms six new chemical bonds in a controlled and ordered process. Two general mechanisms have been suggested for the temporal and spatial control of these transformations. In the spatial positioning model, leader peptide binding promotes certain reactions by establishing the spatial orientation of the substrate peptide relative to the synthetase active sites. In the intermediate structure model, the LctM-catalyzed dehydration and cyclization reactions that occur in two distinct active sites orchestrate the overall process by imparting specific structure into the maturing peptide that facilitates the ensuing reaction. Using isotopically labeled LctA analogues with engineered lacticin 481 biosynthetic machinery and mass spectrometry analysis, we show here that the LctA leader peptide plays critical roles in establishing the modification order and enhancing the catalytic efficiency and fidelity of the synthetase. The data are most consistent with a mechanistic model for LctM where both spatial positioning and intermediate structure contribute to efficient biosynthesis.