Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model.