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

Rocco Moretti, Jon S. Thorson

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)251-258
Number of pages8
JournalAnalytical Biochemistry
Volume377
Issue number2
DOIs
StatePublished - Jun 15 2008

Bibliographical note

Funding Information:
This work was supported, in part, by National Institutes of Health grants GM70637 and U19 CA113297 (to J.S.T.). R.M. was a National Institutes of Health Molecular Bioscience Training Grant trainee (GM07215). J.S.T. is an H. I. Romnes fellow.

Keywords

  • Carbohydrate
  • Enzyme
  • Glycosyltransferase
  • Nucleotide
  • Sugar

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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