Lantibiotics are a group of ribosomally synthesized peptide antibiotics that are post-translationally modified to their bioactive forms. These modifications include initial dehydration of serine and threonine residues followed by cyclization of cysteines onto the dehydro amino acids generated in the first step. Previous in vivo studies revealed that the enzymes responsible for these processes lack absolute substrate specificity. Thus, protein engineering using chemically synthesized unnatural peptide substrates may provide a valuable tool to study structure-function relationships in lantibiotic biosynthesis, and to establish their molecular mechanism(s) of cytotoxicity. In order to achieve these goals, the activities of the enzymes involved in lacticin 481 biosynthesis will be investigated in the first two specific aims. These studies will focus on the interaction of the enzyme with their substrates and the mechanism of dehydration and cyclization. Genetic protein engineering is limited to the 21 physiological amino acids. However, since the size of the prepeptides of lantibiotics is well within the limit of solid phase peptide synthesis, the pool of available amino acids that can be used for "chemical protein engineering" is increased dramatically. Thus, in the fourth specific aim, the natural peptide substrates for post-translational modification will be altered at specific positions by substitution with synthetic unnatural amino acid analogs. This approach may be very powerful to gain insight into the mechanism of biosynthesis of the lantibiotics. Moreover, these studies may produce novel variants of the natural lantibiotics with potentially interesting biological activities. [unreadable] [unreadable]