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

Rocco Moretti; Jon S. Thorson

doi:10.1016/j.ab.2008.03.018

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

Rocco Moretti, Jon S. Thorson

Research output: Contribution to journal › Article › peer-review

71 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 language	English
Pages (from-to)	251-258
Number of pages	8
Journal	Analytical Biochemistry
Volume	377
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2008.03.018
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
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

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10.1016/j.ab.2008.03.018

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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.",

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N2 - 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.

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

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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