The Synthetic Study Of 1-deoxy-D-xylulose 5-phosphate Allowing Deuterium Labelling

1-deoxy-D-xylulose 5-phosphate(DXP) has been identified as a precursor in three major metabolic pathways:the biosynthesis of thiamine diphosphate(vitamin B1,ThPP) and pyridoxol phosphate(vitamin B6) in bacteria and the formation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP) via the 2-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in most bacteria and in the chloroplasts of phototrophic organisms.DXP is synthesized by the DXP synthase(DXS),a ThPP dependent enzyme,catalyzing the condensation of pyruvate and D-glyceraldehyde 3-phosphate.Also, DXP is the substrate for the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase(DXR) which is a rate limiting enzyme in MEP pathway and a potential target for the screening of antibacterial drugs.It converts DXP into the branched compound MEP.In order to study the mechanism of DXR,we required a preparation of active DXS by molecular biological techniques and a flexible synthesis of the substrate which would allow the incorporation of deuterium labeles.In order to obtain purified DXS,the recombinant plasmid pFMH30 was transformed into E. coli BL21(DE3) competent cells,and the DXS protein induced by IPTG was examined by SDS-PAGE analysis.The result of SDS-PAGE showed that the molecular weight of fusion protein DXS with a His6 tag was same as the expected value,approximately 66kDa.The recombinant DXS protein was purified using the(NH₄)₂SO₄ precipitation and Ni²⁺ affinity chromatography,and its concentration was 11.8 mg/ml,determined by Bradford reagent with purity over 90%.The activity test by Reversed-phase ion pair HPLC indicated that this fusion protein can be used for the enzymic synthesis of DXP.DXP,a key metabolite in the MEP pathway for isoprenoid biosynthesis,is conveniently synthesized from pyruvate and D-Glyceraldehyde-3-phosphate(D-GAP) or dihydroxyacetone phosphate(DHAP) with enzymic method,and the synthetic conditions were optimized. D-GAP,an important substrate for the synthesis of DXP,was synthesized from D-fructose-6-phosphate with chemical method and purified with a yield about 60%and more than 80%purity.In D₂O,D-GAP,DHAP or pyruvate can be labled with deuterium in suitable conditions,respectively.Using these labled substrates,we studied the enzymic methods for the synthesis of DXP allowing deuterium labelling in specific position(s) and obtained [3,4-²H₂]DXP,[4-²H]DXP,[3-²H]DXP and[1-²H₃]DXP in higher yield than in chemical procedures reported.These enzymic methods are convenient and flexible and also good for the preparation of ²H-labeled DXP.