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-deoxy-d-xylulose 5-phosphate synthase (DXS) activity based on the reaction of 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 concentration of DXS in the reaction mixture, and (c) the increase in fluorescence intensity correlated (r = 0.99; P < 0.002) with the amount of DX5P formed in the reaction mixture determined radiometrically. The sensitivity of the fluorometric assay is similar to that of the previously described radiometric methods. This assay can be useful for the functional characterization of DXS as well as for the screening of DXS inhibitors with potential antibiotic, herbicidal, or antimalarial action.
Querol
J
J
Departament de Bioquimica i Biologia Molecular, Facultat de Quimica, Universitat de Barcelona, Marti Franquès, 1, Barcelona, 08028, Spain.
Besumbes
O
O
Lois
L M
LM
Boronat
A
A
Imperial
S
S
D062365:Aminobenzoates / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005470:Fluorometry / Q000379:methods*; D005632:Fructose / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D011798:Quinaldines / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D062365:Aminobenzoates; D011798:Quinaldines; D011994:Recombinant Proteins; C017856:3,5-diaminobenzoic acid; D005632:Fructose; C028057:1-deoxyfructose; C037073:2-methylquinoline; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase