Online pyrophosphate assay for analyzing adenylation domains of nonribosomal peptide synthetases

Tiia Kittila, Melanie Schoppet, Max J. Cryle

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.
Original languageEnglish
Pages (from-to)576-584
Number of pages9
JournalChemBioChem
Volume17
Issue number7
DOIs
Publication statusPublished - 1 Apr 2016

Keywords

  • adenylation domain
  • antibiotics
  • enzymes
  • nonribosomal peptide synthetase
  • pyrophosphate detection
  • teicoplanin

Cite this

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title = "Online pyrophosphate assay for analyzing adenylation domains of nonribosomal peptide synthetases",
abstract = "Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.",
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Online pyrophosphate assay for analyzing adenylation domains of nonribosomal peptide synthetases. / Kittila, Tiia; Schoppet, Melanie; Cryle, Max J.

In: ChemBioChem, Vol. 17, No. 7, 01.04.2016, p. 576-584.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Online pyrophosphate assay for analyzing adenylation domains of nonribosomal peptide synthetases

AU - Kittila, Tiia

AU - Schoppet, Melanie

AU - Cryle, Max J.

PY - 2016/4/1

Y1 - 2016/4/1

N2 - Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.

AB - Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.

KW - adenylation domain

KW - antibiotics

KW - enzymes

KW - nonribosomal peptide synthetase

KW - pyrophosphate detection

KW - teicoplanin

UR - http://www.ncbi.nlm.nih.gov/pubmed/26751610

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DO - 10.1002/cbic.201500555

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SN - 1439-4227

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