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Engineering Ribonucleoside Triphosphate Specificity in a Thymidylyltransferase

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journal contribution
posted on 19.08.2008, 00:00 by David L. Jakeman, Jessica L. Young, Malcolm P. Huestis, Pauline Peltier, Richard Daniellou, Caroline Nugier-Chauvin, Vincent Ferrières
Nature’s glycosylation catalysts, glycosyltransferases, indirectly manipulate and control many important biological processes by transferring sugar nucleotide donors onto acceptors. Challenging chemical synthesis impedes synthetic access to sugar nucleotides and limits the study of many glycosyltransferases. Enzymatic access to sugar nucleotides is a rapidly expanding avenue of research, limited only by the substrate specificity of the enzyme. We have explored the promiscuous thymidylyltransferase from Streptococcus pneumoniae, Cps2L, and enhanced its uridylyltransferase and guanidyltransferase activities by active site engineering. Mutagenesis at position Q24 resulted in a variant with 10-, 3-, and 2-fold enhancement of UDP-glucosamine, UDP-mannose, and UDP-N-acetylglucosamine production, respectively. New catalytic activities were observed for the Cps2L variant over the wild-type enzyme, including the formation of GDP-mannose. The variant was evaluated as a catalyst for the formation of a series of dTDP- and UDP-furanoses and notably produced dTDP-Galf in 90% yield and UDP-Araf in 30% yield after 12 h. A series of 3-O-alkylglucose 1-phosphates were also evaluated as substrates, and notable conversions to UDP-3-O-methylglucose and UDP-3-O-dodecylglucose were achieved with the variant but not the wild-type enzyme. The Q24S variant also enhanced essentially all thymidylyltransferase activities relative to the wild-type enzyme. Comparison of active sites of uridylyltransferases and thymidylyltransferases with products bound indicate the Q24S variant to be a new approach in broadening nucleotidylyltransferase activity.