A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.
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.
Moretti
Rocco
R
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Thorson
Jon S
JS
D002851:Chromatography, High Pressure Liquid; D005958:Glucosephosphates / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism*; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000201:enzymology*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*
D005958:Glucosephosphates; D006027:Glycosides; D062385:Hydroxybenzoates; D013403:Sugar Phosphates; C002429:4-hydroxybenzoic acid hydrazide; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases