Identification And Characterization Of A O-methyltransferase In A Lassomycin-like Lasso Peptide Biosynthetic Pathway

Lassomycin is a newly discovered lasso peptide with a unique lasso folding structure,which has high anti-TB activity against multidrug-resistant Mycobacterium tuberculosis,so it has high reaserch study.In this experiment,two novel lassomycin-like lasso peptide biosynthesis pathways were identified and it is the first time a novel C-terminal peptide carboxyl O-methyltransferase involved in these pathways was characterized.This thesis contains that:1.The target protein stspM was successfully obtained by molecular biological methods,and the study of precursor peptide methylation were carried out later.The results revealed that this new methyltransferase could specifically methylate the C-terminal of precursor peptide which containing 37 amino acid residues,eventually leading to lassomycin-like C-terminal methylated lasso peptides.2.Substrate specificity studies have shown that stspM could methylate the C-terminal of diverse peptides with different lengths and sequences,the last residue is also not essential for catalysis,which make stspM a universal catalyst for peptide C-terminal protection and activation.3.The investigation of the stspM biochemical characterization show that the optimal temperature was 50℃ and the optimal pH was 6.0.Under optimal conditions,the apparent Km of 81.8±5.6 μM and a kcat of 0.67±0.03 min-1 were determined.4.To validate the catalytic center of stspM,His242 and Glu296 were mutated to Ala by SLIM mutagenesis,purified and then assayed with stspA.As expected,these two mutants completely lost activity.The results demonstrated that they are indeed the active center for stspM.5.Bioinformatic analysis of lasso peptide biosynthetic gene clusters featuring methyltransferase shown that the methyltransferase-containing gene clusters were very close to the lariatin system,indicating that they are originally from the same ancestor.