Mechanistic Evaluation of a Nucleoside Tetraphosphate with a Thymidylyltransferase

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5′-tetraphosphate (p₄dT) was evaluated with a thymidylyltransferase, Cps2L. We have identified p₄dT as a substrate for Cps2L and evaluated the reaction pathway by analysis of products using high-performance liquid chromatography, liquid chromatography and tandem mass spectrometry, and ³¹P nuclear magnetic resonance spectroscopy. Product analysis confirmed production of dTDP-Glc and triphosphate (P₃) and showed no trace of dTTP-Glc and PP_i, which could arise from alternative pathways for the reaction mechanism