Characterization of a methyltransferase for iterative N-methylation at the leucinostatin termini in Purpureocillium lilacinum

Abstract N -methyltransferase (NMT)-catalyzed methylation at the termini of nonribosomal peptides (NRPs) has rarely been reported. Here, we discover a fungal NMT LcsG for the iterative terminal N -methylation of a family of NRPs, leucinostatins. Gene deletion results suggest that LcsG is essential for leucinostatins methylation. Results from in vitro assays and HRESI-MS-MS analysis reveal the methylation sites as NH 2 , NHCH 3 and N(CH 3 ) 2 in the C-terminus of various leucinostatins. LcsG catalysis yields new lipopeptides, some of which demonstrate effective antibiotic properties against the human pathogen Cryptococcus neoformans and the plant pathogen Phytophthora infestans . Multiple sequence alignments and site-directed mutagenesis of LcsG indicate the presence of a highly conserved SAM-binding pocket, along with two possible active site residues (D368 and D395). Molecular dynamics simulations show that the targeted N can dock between these two residues. Thus, this study suggests a method for increasing the variety of natural bioactivity of NPRs and a possible catalytic mechanism underlying the N -methylation of NRPs.