Lantibiotic engineering: peptide engineering approach using the modification enzymes

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Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. Ribosomally synthesized precursor peptide, NukA undergoes introduction of unusual amino acids by a single modification enzyme, NukM, and the leader sequence is removed and the mature peptide is secreted by ABC transporter NukT. NukM was divided into two domains, NukMN and NukMC, based on a prediction that each domain is responsible for dehydration (phosphorylation and further phosphate elimination) and cyclization, respectively. The in vitro reconstitution of NukM, NukMN, and NukMC revealed that NukM can fully modify NukA, and NukMN could partially phosphorylate and dehydrate it. A series of NukT mutants were constructed and their transport activity in vivo and peptidase activity in vitro were investigated. It was suggested that peptidase activity of NukT depends on ATP hydrolysis, and the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain.