Journal
CHEMISTRY & BIOLOGY
Volume 15, Issue 4, Pages 393-401Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2008.02.017
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Funding
- NCI NIH HHS [U19 CA113297-030002, U19 CA113297, U19 CA113297-050002, U19 CA113297-010002, U19 CA113297-020002, U19 CA113297-040002] Funding Source: Medline
- NIAID NIH HHS [R01 AI052218-06, R01 AI052218-07, R01 AI052218-08, R01 AI052218, AI52218, R37 AI052218] Funding Source: Medline
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A comprehensive two-phase hot spot saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-through put screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.
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