The dehydratase NisB performs stepwise tRNA(Glu)-dependent glutamylation of Ser/Thr residues and subsequent glutamate elimination to effect eight dehydrations in the biosynthesis of the antibacterial peptide nisin. Its substrate, NisA, bears a C-terminal core peptide that is modified and an N-terminal leader peptide (LP) that is not modified but that is required for efficient dehydration. To elucidate the mechanism of LP-NisB interactions during dehydration, we engineered a disulfide that covalently links the NisA LP to NisB. The enzyme fully dehydrated tethered NisA, confirming the functional LP binding site and supporting a mechanism where NisB uses a single LP binding site for glutamylation and elimination. We also show an order of NisA and tRNA(Glu) binding to NisB that enables dehydration.