A Capture Strategy for the Identification of Thio-Templated Metabolites. Academic Article uri icon


  • Nonribosomal peptide synthetase and polyketide synthase systems are home to complex enzymology and produce compounds of great therapeutic value. Despite this, they have continued to be difficult to characterize due to their substrates remaining enzyme-bound by a thioester bond. Here, we have developed a strategy to directly trap and characterize the thioester-bound enzyme intermediates and applied the strategy to the azinomycin biosynthetic pathway. The approach was initially applied in vitro to evaluate its efficacy and subsequently moved to an in situ system, where a protein of interest was isolated from the native organism to avoid needing to supply substrates. When the nonribosomal peptide synthetase AziA3 was isolated from Streptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validated in vitro with a nonribosomal peptide synthetase involved in colibactin biosynthesis. In the long term, this method will be utilized to characterize thioester-bound metabolites within not only the azinomycin biosynthetic pathway but also other cryptic metabolite pathways.

published proceedings

  • ACS Chem Biol

altmetric score

  • 3.5

author list (cited authors)

  • Washburn, L. A., Nepal, K. K., & Watanabe, C.

citation count

  • 1

complete list of authors

  • Washburn, Lauren A||Nepal, Keshav K||Watanabe, Coran MH

publication date

  • September 2021