Unimodular Methylation by Adenylation-Thiolation Domains Containing an Embedded Methyltransferase

J Mol Biol. 2020 Oct 2;432(21):5802-5808. doi: 10.1016/j.jmb.2020.09.004. Epub 2020 Sep 11.


Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.

Keywords: biosynthesis; enzyme mechanism; interrupted adenylation domain; natural product; nonribosomal peptide synthetase.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Monophosphate / metabolism
  • Depsipeptides / chemistry
  • Depsipeptides / metabolism*
  • Echinomycin / chemistry
  • Echinomycin / metabolism*
  • Kinetics
  • Methylation
  • Methyltransferases / chemistry
  • Methyltransferases / metabolism*
  • Microsporidia / enzymology*
  • Microsporidia / metabolism
  • Peptide Biosynthesis, Nucleic Acid-Independent
  • Peptide Synthases / chemistry
  • Peptide Synthases / metabolism*
  • Protein Domains
  • Streptomyces / enzymology*
  • Streptomyces / metabolism
  • Sulfhydryl Compounds / metabolism


  • Depsipeptides
  • Sulfhydryl Compounds
  • thiocoraline
  • Adenosine Monophosphate
  • Echinomycin
  • Methyltransferases
  • Peptide Synthases
  • non-ribosomal peptide synthase