A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay

Anal Biochem. 2008 Jun 15;377(2):251-8. doi: 10.1016/j.ab.2008.03.018. Epub 2008 Mar 15.


A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, High Pressure Liquid
  • Glucosephosphates / metabolism
  • Glycosides / metabolism
  • Hydroxybenzoates / metabolism
  • Nucleotidyltransferases / metabolism*
  • Reproducibility of Results
  • Salmonella enterica / enzymology*
  • Substrate Specificity
  • Sugar Phosphates / metabolism*


  • Glucosephosphates
  • Glycosides
  • Hydroxybenzoates
  • Sugar Phosphates
  • 4-hydroxybenzoic acid hydrazide
  • glucose-1-phosphate
  • Nucleotidyltransferases