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Active site mapping of iterative polyketide synthases and the detection of polyketide intermediates using high- resolution Fourier Transform mass spectrometry

  • Author(s): Meehan, Michael Joseph
  • et al.
Abstract

In an attempt to better understand the biosynthes of the enediyne core of the antitumor compound C-1027 by SgcE and to investigated the iterative steps involved in the formation of the methylbutyrl side-chain of lovastatin by LovF, high resolution mass spectrometry was used to identify peptides containing domain active sites of these polyketide synthases and to determine with very high mass accuracy the mass of substrates covalently attached to the active site. In particular the phosphopantetheinyl ejection assay was used to successfully detect the formation of the methylbutyrate by LovF on the ACP active site. It was found that methylbutyrate is detextable only at very low substrate concentrations, but complete formation of the product was carried out by LovF. SgcE was found to be active and capable of catalyzing elongation of a polyene intermediate. The substrate loading and one iteration of elongation was detected using mass spectrometry and the phosphopantetheinyl ejection assay. The results from these experiments showed the power of high-resolution mass spectrometry in studying the mechanisms of action by which these iterative polyketide synthases function and form their fully elongated products.

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