ABSTRACT
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.
Subject(s)
Diphosphonates/chemistry , Diphosphonates/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nucleoside-Triphosphatase/chemistry , Nucleoside-Triphosphatase/metabolism , Thymidine/chemistry , Binding Sites , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Models, Molecular , Nucleoside-Triphosphatase/genetics , Protein Engineering , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/genetics , Substrate Specificity , Thymidine/geneticsABSTRACT
Synthetic methods were investigated for the preparation of O and S-glucosyl thiophosphates and glucosyl 1C-thiophosphonate. Four protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose 1-thiophosphate. The effect of various reaction conditions and the nature of the carbohydrate and thiophosphate protecting groups and how they impact both the yields and α/ß diastereoselectivity of the glucosyl thiophosphate products were explored. A novel isomerization from an O-linked to S-linked glucosyl thiophosphate was observed. α-D-Glucose-1C-thiophosphonate was synthesized and evaluated as a substrate for the thymidylyltransferase, Cps2L. Tandem mass spectrometric analysis determined the position of sulfur in the sugar nucleotide product.
Subject(s)
Glucose/analogs & derivatives , Glucosephosphates/chemistry , Glucosephosphates/metabolism , Nucleotidyltransferases/metabolism , Phosphates/metabolism , Thymine Nucleotides/biosynthesis , Thymine Nucleotides/metabolism , Carbohydrate Conformation , Enzyme Activation , Glucose/biosynthesis , Glucose/chemistry , Glucose/metabolism , Glucosephosphates/chemical synthesis , Phosphates/chemistry , Tandem Mass Spectrometry , Thymine Nucleotides/chemistryABSTRACT
Efficient enzymatic syntheses of isosteric phosphono analogues of sugar nucleotides have been accomplished using a thymidylyltransferase.
