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Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.

ABSTRACT: 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-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.

SUBMITTER: Williams GJ

PROVIDER: S-EPMC2813856 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.

Williams Gavin J GJ Goff Randal D RD Zhang Changsheng C Thorson Jon S JS

Chemistry & biology 20080401 4

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-throughput screen to provid ...[more]

PMID: 18420146

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