Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.
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.
Williams
Gavin J
GJ
Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, National Cooperative Drug Discovery Program, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705, USA.
Goff
Randal D
RD
Zhang
Changsheng
C
Thorson
Jon S
JS
D019943:Amino Acid Substitution; D000596:Amino Acids; D001665:Binding Sites; D002384:Catalysis; D019020:Directed Molecular Evolution; D005453:Fluorescence; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009675:Novobiocin / Q000378:metabolism*; D009827:Oleandomycin / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D009675:Novobiocin; D016695:Glycosyltransferases; D009827:Oleandomycin