Assay for GTP Cyclohydrolase II Activity in Bacterial Extracts

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.