Thymidylyltransferases (thymidine diphospho pyrophosphorylases) are nucleotidylyltransferases that play key roles in the biosynthesis of carbohydrate components within bacterial cell walls and in the biosynthesis of glycosylated natural products. They catalyze the formation of sugar nucleotides concomitant with the release of pyrophosphate. Protein engineering of thymidylyltransferases has been an approach for the production of a variety of non-physiological sugar nucleotides. In this work, we have explored chemical approaches towards modifying the activity of the thymidylyltransferase (Cps2L) cloned from S. pneumoniae, through the use of chemically synthesized 'activated' nucleoside triphosphates with enhanced leaving groups, or by switching the metal ion co-factor specificity. Within a series of phosphonate-containing nucleoside triphosphate analogues, thymidylyltransferase activity is enhanced based on the acidity of the leaving group and a Brønsted-type analysis indicated that leaving group departure is rate limiting. We have also determined IC50 values for a series of bisphosphonates as inhibitors of thymidylyltransferases. No correlation between the acidity of the inhibitors (pKa) and the magnitude of enzyme inhibition was found.
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