Substrate Specificity of the Secreted Nisin Leader Peptidase NisP.

Abstract

Nisin (NisA) is an antimicrobial peptide produced by Lactococcus lactis and belongs to the class of lanthipeptides, more specifically to the class of lantibiotics. They are ribosomally synthesized as a precursor peptide and are comprised of an N-terminal leader peptide and a C-terminal core peptide. The core peptide is post-translationally modified and contains dehydrated amino acids in addition to five (methyl)-lanthionine rings, which are crucial for its activity. The leader peptide serves as a signal sequence and ensures that NisA remains inactive but secretion-competent within the cell. After translocation into the extracellular space, the leader peptide is cleaved by the leader peptidase NisP, resulting in active nisin. NisP is an extracellular subtilisin-like serine protease, which recognizes the cleavage site GASPR|IT located at the C-terminal end of the leader peptide. Here, we present the biochemical characterization of secreted and purified NisP (NisPs) with its natural substrate, the fully modified NisA (mNisA). Furthermore, we determined the kinetic parameters of NisPs in the presence of NisA containing different modification states. Additionally, in vitro data revealed that NisPs can efficiently cleave the leader peptide of mNisA. However, it is strictly dependent on the modification state of the core peptide. Thus, NisPs has a sequence-based cleavage activity, and the presence of at least one lanthionine ring is crucial for optimal substrate recognition and subsequent cleavage.