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 language | English |
|---|---|
| Pages (from-to) | 845-851 |
| Number of pages | 7 |
| Journal | Journal of Biomolecular Screening |
| Volume | 16 |
| Issue number | 8 |
| DOIs | |
| State | Published - 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