Effects of cleavage sites of leader peptides on the activities of class Ⅱ lantibiotic peptidases

Abstract:

[Background] Class Ⅱ lantibiotics are ribosomally synthesized and post-translationally modified peptides, mainly produced by Gram-positive bacteria. In the last step of its biosynthesis, the N-terminal peptidase domain of transporter protein LanT cleaves the leader peptide to produce active lantibiotics. However, the removal mechanism of leader peptide in this class lantibiotics is still not clear. [Objective] To investigate the effects of cleavage sites on the activity of peptidases domain BovT150 and SboT150 from different streptococci. [Methods] Expression vectors for the precursor peptides with mutated cleavage sites were constructed by site-directed ligase-independent mutagenesis and then the wild-type precursors (BovAm and SboAm), their mutant precursors, as well as the corresponding peptidases (BovT150 and SboT150) were expressed and purified in E. coli. The precursors were separately incubated with each peptidase in vitro and the removal efficiencies of the leader peptides were assessed by HPLC, antimicrobial activity assay and MALDI-TOF MS. [Results] Both cleavage sites GG and GA of BovAm and SboAm allowed BovT150 to retain peptidase activity, and Gly was more suitable to be processed by BovT150. Only cleavage sites GG and GA of SboAm were accessible to SboT150, which cleaved Ala more efficiently. [Conclusion] The change of amino acid residues at the cleavage sites of leader peptides affected the efficiencies of class Ⅱ lantibiotic peptidases in varying degrees.