Biosynthesis of the (2S,3R)-3-methyl glutamate residue of nonribosomal lipopeptides.
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.
Milne
Claire
C
School of Chemistry and Department of Biomolecular Sciences, The University of Manchester, P.O. Box 88, Manchester M60 1QD, United Kingdom.
Powell
Amanda
A
Jim
John
J
Al Nakeeb
Majid
M
Smith
Colin P
CP
Micklefield
Jason
J
D002853:Chromatography, Liquid; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D008074:Lipoproteins / Q000138:chemical synthesis*; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000138:chemical synthesis*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism
C425779:3-methylglutamic acid; D008074:Lipoproteins; D010455:Peptides; D018698:Glutamic Acid