Biosynthesis of the(2S,3R)-3-Methyl Glutamate Residue of Nonribosomal Lipopeptides

摘要

The calcium-dependent antibiotics(CDAs)and daptomycin are therapeutically relevant nonribosomal lipopeptide antibiotics that contain penultimate C-terminal 3-methyl glutamate(3-MeGlu)residues.Comparison with synthetic standards showed that(2S,3R)-configured 3-MeGlu is present in both CDA and daptomycin.Deletion of a putative methyltransferase gene glmT from the cda biosynthetic gene cluster abolished the incorporation of 3-MeGlu and resulted in the production of Glu-containing CDA exclusively.However,the 3-MeGlu chemotype could be re-established through feeding synthetic 3-methyl-2-oxoglutarate and(2S,3R)-3-MeGlu,but not(2S,3S)-3-MeGlu.This indicates that methylation occurs before peptide assembly,and that the module 10 A-domain of the CDA peptide synthetase is specific for the(2S,3R)-stereoisomer.Further mechanistic analyses suggest that GlmT catalyzes the SAM-dependent methylation of alpha-ketoglutarate to give(3R)-methyl-2-oxoglutarate,which is transaminated to(2S,3R)-3-MeGlu.These insights will facilitate future efforts to engineer lipopeptides with modified glutamate residues,which may have improved bioactivity and/or reduced toxicity.