A High-Throughput Screen for Directed Evolution of the Natural Product Sulfotransferase LipB

作者：Koryakina Irina; Neville Jessica; Nonaka Koichi; Van Lanen Steven G; Williams Gavin J^*

来源：Journal of Biomolecular Screening, 2011, 16(8): 845-851.

DOI：10.1177/1087057111413273

• chemistry
• medicinal
• organic
• synthetic
• and combinatorial
• enzyme assays or enzyme kinetics
• anti-infective drugs
• natural products screening

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. (Journal of Biomolecular Screening. 2011;16:845-851)

• 出版日期2011-9