A high-throughput screen for directed evolution of the natural product sulfotransferase LipB

Irina Koryakina, Jessica Neville, Koichi Nonaka, Steven G. Van Lanen, Gavin J. Williams

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.

Original languageEnglish
Pages (from-to)845-851
Number of pages7
JournalJournal of Biomolecular Screening
Volume16
Issue number8
DOIs
StatePublished - Sep 2011

Keywords

  • And combinatorial
  • Anti-infective drugs
  • Chemistry
  • Enzyme assays or enzyme kinetics
  • Medicinal
  • Natural products screening
  • Organic
  • Synthetic

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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