An enhanced chemoenzymatic method for loading substrates onto carrier protein domains

Tiia Kittilä, Max J. Cryle

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Non-ribosomal peptide synthetase (NRPS) machineries produce many medically relevant peptides that cannot be easily accessed by chemical synthesis. Thus, understanding NRPS mechanism is of crucial importance to allow efficient redesign of these machineries to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto PCP domains have been described, but all suffer from difficulties in either the complexity of chemical synthesis or low enzymatic efficiency. Here, we describe an enhanced chemoenzymatic loading method that combines 2 approaches into a single, highly efficient one-pot loading reaction. First, d-pantetheine and ATP are converted into dephospho-coenzyme A via the actions of 2 enzymes from coenzyme A (CoA) biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which is inhibited by phosphoadenylates. This route does not depend on activity of the commonly problematic dephospho-CoA kinase and, therefore, offers an improved method for substrate loading onto PCP domains.

Original languageEnglish
Pages (from-to)372-379
Number of pages8
JournalBiochemistry and Cell Biology
Volume96
Issue number3
DOIs
Publication statusPublished - 1 Jan 2018

Keywords

  • Carrier protein
  • Coenzyme A
  • Non-ribosomal peptide synthetase
  • Phosphopantetheinyl transferase
  • Post-translational modification

Cite this

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An enhanced chemoenzymatic method for loading substrates onto carrier protein domains. / Kittilä, Tiia; Cryle, Max J.

In: Biochemistry and Cell Biology, Vol. 96, No. 3, 01.01.2018, p. 372-379.

Research output: Contribution to journalArticleResearchpeer-review

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