A Fluorometric Assay for the Determination of 1-Deoxy-D-xylulose 5-Phosphate Synthase Activity Jordi Querol, Oscar Besumbes, Luisa Maria Lois, Albert Boronat, and Santiago Imperial 1 Departament de Bioquimica i Biologia Molecular, Facultat de Quimica, Universitat de Barcelona, Marti Franque `s, 1, 08028 Barcelona, Spain Received March 20, 2001; published online August 2, 2001 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-de- oxy-D-xylulose 5-phosphate (DX5P) with 3,5-diamino- benzoic acid in an acidic medium to form a highly fluorescent quinaldine derivative. The assay was vali- dated in three ways: (a) for a fixed amount of DXS in the reaction mixture the emitted fluorescence in- creased 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. © 2001 Academic Press Key Words: 1-deoxy-D-xylulose 5-phosphate synthase; fluorometric assay; 3,5-diaminobenzoic acid; isopre- noid biosynthesis. Isoprenoids represent the largest group of natural products found in nature with more than 30,000 differ- ent compounds identified so far (1). In spite of the great diversity of structures and functions, all isoprenoids are synthesized from a common precursor, isopentenyl diphosphate (IPP), 2 and its allylic isomer DMAPP (2). In heterotrophic bacteria and plastids (3–7) IPP is syn- thesized by a mevalonate-independent pathway known as the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (8 –10). The first reaction of the MEP pathway is the synthesis of 1-deoxy-D-xylulose 5-phosphate (DX5P) by condensation of (hydroxyethyl)thiamin de- rived from the decarboxylation of pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate (4, 5) (Fig. 1). This reaction is catalyzed by the enzyme 1-de- oxy-D-xylulose 5-phosphate synthase (DXS), a novel type of transketolase encoded by the dxs gene in Esch- erichia coli (11, 12). In addition to its role as the first intermediate in the synthesis of IPP, DX5P is also a precursor for the synthesis of pyridoxol (vitamin B6) and thiamin (vitamin B1) in bacteria and plants (13, 14). The location of DXS at a branch point of three essential biosynthetic pathways in bacteria and higher plants, together with the absence of this enzyme in animals, makes DXS an attractive target for the design of drugs with potential applications as antibiotics and herbicides (8, 10). The recent report of the occurrence and essentiality of the MEP pathway in the parasite Plasmodium falciparum extends the interest of DXS inhibitors to the development of antimalarial drugs (15). Strategies for the systematic screening of DXS inhib- itors should rely on the availability of a simple, sensi- tive, and nonhazardous assay well suited to automa- tion. The DXS assays most widely used rely on radiochemical methods using [2- 14 C]pyruvate (6, 11, 12, 16). Like most radiochemical assays they have the disadvantage that the labeled product must be sepa- rated from the labeled substrate at the end of the incubation and this demands additional manipula- 1 To whom correspondence should be addressed. Fax: (+34) 93- 4021219. E-mail: imperial@bq.ub.es. 2 Abbreviations used: DABA, 3,5-diaminobenzoic acid; DX, 1-deoxy-D-xylulose; DX5P, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy- D-xylulose 5-phosphate synthase; IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; SDS, sodium dodecyl sulfate; TLC, thin-layer chromatography. 0003-2697/01 $35.00 101 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 296, 101–105 (2001) doi:10.1006/abio.2001.5234, available online at http://www.idealibrary.com on