Engineering Bifunctional Enzymes Capable of Adenylating and Selectively Methylating the Side Chain or Core of Amino Acids

Taylor A. Lundy, Shogo Mori, Sylvie Garneau-Tsodikova

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Nonribosomal peptides (NRPs) are known sources of therapeutics. Some nonribosomal peptide synthetase assembly lines contain unique functional interrupted adenylation (A) domains, where nature has combined two different functional domains into one bifunctional enzyme. Most often these interrupted A domains contain a part of a methylation (M) domain embedded in their sequence. Herein, we aimed to emulate nature and create fully functional interrupted A domains by inserting two different noncognate M domains, KtzH(MH) and TioS(M3S), into a naturally occurring uninterrupted A domain, Ecm6(A1T1). We evaluated the engineered enzymes, Ecm6(A1aMHA1bT1) and Ecm6(A1aM3SA1bT1), by a series of radiometric assays and found that not only do they maintain A domain activity, but also they gain the site-specific methylation patterns observed in the parent M domain donors. These findings provide an exciting proof-of-concept for generating interrupted A domains as future tools to modify NRPs and increase the diversity and activity of potential therapeutics.

Original languageEnglish (US)
Pages (from-to)399-404
Number of pages6
JournalACS Synthetic Biology
Volume7
Issue number2
DOIs
StatePublished - Feb 16 2018
Externally publishedYes

Keywords

  • echinomycin
  • kutznerides
  • natural products
  • nonribosomal peptide biosynthesis
  • synthetic biology
  • thiocoraline

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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