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...

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Bibliographic Details
Published in:Analytical biochemistry 2001-09, Vol.296 (1), p.101-105
Main Authors: Querol, J, Besumbes, O, Lois, L M, Boronat, A, Imperial, S
Format: Article
Language:English
Subjects:
Aminobenzoates - analysis
Aminobenzoates - chemistry
Escherichia coli - enzymology
Fluorometry - methods
Fructose - analogs & derivatives
Fructose - analysis
Fructose - chemistry
Quinaldines - analysis
Quinaldines - chemistry
Recombinant Proteins - analysis
Sensitivity and Specificity
Transferases - isolation & purification
Transferases - metabolism
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Summary: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.
ISSN:0003-2697
DOI:10.1006/abio.2001.5234