Stereospecific Enzymatic Transformation of α-Ketoglutarate to (2S,3R)-3-Methyl Glutamate during Acidic Lipopeptide Biosynthesis

Abstract

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.

Genomics and the ancient origins of the daptomycin biosynthetic gene cluster

“…The dptI gene encodes a methyltransferase involved in the formation of 3-methyl-glutamic acid (3mGlu) from a-ketoglutarate. 15,28 The DptI protein has only two distantly related functional homologs, LptI 29,30 from the lipopeptide A54145 pathway and GlmT 28,31 from the calcium-dependent antibiotic pathway. DptI shows only 37 and 36% amino acid identities to LptI and GlmT, respectively.…”

Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A

“…The 20-ORF tar locus maintains the dpt genes that encode the three NRPS megasynthetases. Furthermore, the enzymes required for biosynthesis of the nonproteinogenic amino acid residues 3-methylglutamic acid (3mGlu) (38,39) …”

Paradigm Shifts in Fungal Secondary Metabolite Research: Unusual Fatty Acids Incorporated into Fungal Peptides

“…The acidic lipopeptides, including the calciumdependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species (Vilhena et al, 2012;Baltz et al, 2005). Calcium-dependent antibiotics, CDA1b, CDA2a, CDA2b, CDA3a, CDA3b, CDA4a and CDA4b contain 3-pentyloxirane-2-carboxylic acid [144], and CDA1 and CDA2 contain 3-propyloxirane-2-carboxylic acid [145] (Mahlert et al, 2007).…”