title: Mechanistic evaluation of a nucleoside tetraphosphate with a thymidylyltransferase.
abstract: 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 PPi, which could arise from alternative pathways for the reaction mechanism.
authors: Department of Chemistry, Dalhousie University , P.O. Box 15000, Halifax, Canada B3H 4R2.
authors: Stephanie M
authors: SM
authors: Forget
authors: Deborah A
authors: DA
authors: Smithen
authors: Alison
authors: A
authors: Jee
authors: David L
authors: DL
authors: Jakeman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011071:Poly T / Q000737:chemistry* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011071:Poly T; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase