A fluorometric assay for the determination of 1-deoxy-D-xylulose 5-phosphate synthase activity

We report a novel fluorometric end-point assay for the determination of 1-d eoxy-D-xylulose 5-phosphate synthase (DXS) activity based on the reaction o f 1-deoxy-D-xylulose 5-phosphate (DX5P) with 3,5-diaminobenzoic acid in an acidic medium to form a highly fluorescent quinaldine derivative. The assay was validated in three ways: (a) for a fixed amount of DXS in the reaction mixture the emitted fluorescence increased linearly with the reaction time , (b) for a fixed reaction time fluorescence intensity increased with the c oncentration of DXS in the reaction mixture, and (c) the increase in fluore scence intensity correlated (r = 0.99; P < 0.002) with the amount of DX5P f ormed in the reaction mixture determined radiometrically. The sensitivity o f the fluorometric assay is similar to that of the previously described rad iometric methods. This assay can be useful for the functional characterizat ion of DXS as well as for the screening of DXS inhibitors with potential an tibiotic, herbicidal, or antimalarial action. (C) 2001 Academic Press.