High-performance liquid chromatography assay for 1-deoxy-D-xylulose 5-phosphate synthase activity using fluorescence detection.
A high-performance liquid chromatography assay for activity of 1-deoxy-D-xylulose 5-phosphate synthase, an early enzyme in the recently discovered 2-C-methyl-D-erythritol-4-phosphate pathway, was developed. In this assay, the enzymatic product 1-deoxy-D-xylulose was first derivatized with a fluorescent reagent 2-anthranilic acid, followed by separation using HPLC on a Nova-Pak phenyl column with a mobile phase containing CH3CN-water-1-butylamine-tetrahydrofuran-H3PO4 (2:97:0.125:0.5:0.25, v/v). The eluate was monitored by fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 425 nm for quantitation of the fluorescent derivative. A linear response was obtained between 5 and 200 ng of 1-deoxy-D-xylulose. This assay was successfully applied to measure the 1-deoxy-D-xylulose 5-phosphate synthase activity in a recombinant E. coli overexpressing dxs gene. It demonstrated that this assay is simple, sensitive and selective compared to the methods used at present.
Han
Ying-Shan
YS
Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Sabbioni
Cesare
C
van der Heijden
Robert
R
Verpoorte
Robert
R
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014166:Transferases / Q000378:metabolism*; D062367:ortho-Aminobenzoates
D005456:Fluorescent Dyes; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase