Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1392131 | Chemistry & Biology | 2008 | 9 Pages |
SummaryA 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-throughput 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.