Lantibiotics are antibacterial
peptides containing unique
thioether cross-links termed
lanthionine and methyllanthionine. NukM, the modifying
enzyme of
nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the
dehydration of specific Ser/Thr residues in a precursor
peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor
peptide of
nisin is modified by 2
enzymes, NisB and NisC, which mediate
dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known
proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate
peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze
dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and
dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification
enzyme.