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Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay

2014; Royal Society of Chemistry; Volume: 13; Issue: 3 Linguagem: Inglês

10.1039/c4ob02057j

1477-0539

Stephanie M. Forget, Alison Jee, Deborah A. Smithen, Rajendra C. Jagdhane, Shazia Anjum, Stephen A. Beaton, David R. J. Palmer, Raymond T. Syvitski, David L. Jakeman,

Glycosylation and Glycoproteins Research

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.