Abstract
Riboflavin is the precursor of flavin nucleotides FMN and FAD, they play significant roles in all organisms. GTP is the initial precursor on riboflavin biosynthesis pathway and GTP cyclohydrolase II catalyzes the first step of this pathway. It converts GTP to 2,5-diamino-6-ribosylamino-4 (3H) -pyrimidinone 5'-phosphate. This protocol provides a reliable and fast method to assay GTP cyclohydrolase II activity from crude bacterial extracts. The product of the reaction catalyzed by GTP cyclohydrolase II, 2,5-diamino-6-ribosylamino-4 (3H) -pyrimidinone 5'-phosphate, is converted to its fluorescent derivative 6,7-dimethylpterin, which is then separated on a XTerra MS C18 column and detected using fluorescence HPLC system.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. HPLC chromatograms of 2.5 µM 6,7-dimethylpterin standard (A), product of GTP cyclohydrolase II assay from bacterial extracts (B) and co-elution of product from GTP cyclohydrolase II assay with 6,7-dimethylpterin standard (C). HPLC mobile phase: 10% methanol and 90% phosphoric acid (v/v). Excitation and emission wavelengths were 330 nm and 435 nm respectively.
Recipes
Acknowledgments
This protocol was adapted from Yurgel et al., 2014. This work was supported by the Agricultural Research Center (WNP-00773) at Washington State University and grant DE-FG0396ER20225 Vol. 27, No. 5, 2014 / 445 from the Energy Biosciences Program at the United States Department of Energy, and by grant NSF-MCB 1052492 to S. Rajamani. This activity was funded, in part, with an Emerging Research Issues Internal Competitive grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences and Biologically-Intensive Agriculture and Organic Farming (BIOAg) Internal Competitive grant from The Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University to S. Yurgel. We thank M. Kahn for discussions and the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support.
References
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