Stereospecific enzymatic transformation of alpha-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis.
The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.
Mahlert
Christoph
C
Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse D-35032 Marburg, Germany.
Kopp
Florian
F
Thirlway
Jenny
J
Micklefield
Jason
J
Marahiel
Mohamed A
MA
D000586:Amination; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis* / Q000138:chemical synthesis; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity