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 language | English |
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Pages (from-to) | 251-258 |
Number of pages | 8 |
Journal | Analytical Biochemistry |
Volume | 377 |
Issue number | 2 |
DOIs | |
State | Published - 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.
Funding
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.
Funders | Funder number |
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National Institutes of Health (NIH) | U19 CA113297 |
National Institute of General Medical Sciences | R01GM070637 |
Keywords
- Carbohydrate
- Enzyme
- Glycosyltransferase
- Nucleotide
- Sugar
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology