SUBSTRATE RECOGNITION AND SPECIFICITY OF NISB, THE LANTIBIOTIC DEHYDRATASE INVOLVED IN NISIN BIOSYNTHESIS

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl)lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified and the dehydrated prenisin binds to NisB with affinities of 1.05 ± 0.25 μM and 0.3 ± 0.07 µM, whereas the fully modified derivatives bind with respective low affinity of 10.5 ± 1.7 μM. The much lower affinity for the fully modified prenisin related to a >20-fold higher off rate. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD-box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.