The deoxyxylulose phosphate (DXP) pathway gains importance beside the established mevalonate pathway. Gram-negative bacteria, some gram-positive bacteria and plants utilize the DXP pathway, but the pathway is not found in mammals. Hence the enzymes and intermediates are attractive targets for the development of novel antibiotic, antimalarial and herbicidal agents. The synthesis of commercially not available intermediates was required for investigation of the biosynthetic steps in the DXP pathway. Coding sequences of the seven enzymes from Escherichia coli were cloned for enzyme-assisted synthesis of metabolites: 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 2C-methyl-D-erythritol 2,4-cyclodiphosphate reduktase (GcpE), 1-hydroxy-2-methylbutenyl 4-diphosphate reduktase (LytB) and D-xylulokinase (XylB) for the phosphorylation of 1-deoxy-D-xylulose. These enzymes and the 1-deoxy-D-xylulose 5-phosphate reduktoisomerase (DXR) were expressed with hexahistidin tag and purified by metal affinity chromatography. The soluble enzymes showed catalytic activity and could be synthesised in milligram amounts. By different enzymatic and chemical syntheses intermediates of the DXP pathway with different isotope-labelling patterns were obtained, e.g. [1,2-14C2]1-deoxy-D-xylulose 5-phosphate und [1,2-13C2]1-deoxy-D-xylulose 5-phosphate. Isopentenyl diphosphate dimethylallyl diphosphate (IDP) isomerase is a nonessential enzyme for isoprenoid synthesis in bacteria. The function of the enzyme in higher plants remained to be elucidated. IDP isomerase was isolated from a cDNA library of Cannabis sativa by homology cloning. The open reading frame included 810 nucleotides and encoded a protein of 269 amino acids. This enzyme and the IDP isomerase from Escherichia coli were cloned with a hexahistidin tag. Protein expression and purification with metal affinity chromatography led to soluble enzymes in milligram amounts. The recombinant IDP isomerases from C. sativa and E. coli showed catalytic activity. In the context of these investigations the published HPLC-method for detection of enzymatic activity of IDP isomerase was checked. Functional analysis of plant IDP isomerase was carried out on Nicotiana benthamiana. By virus induced RNA-interference (virus induced gene silencing) the idi-gene coding for IDP isomerase was silenced. Considerable alterations compared to the control plants were detected macroscopically, microscopically and by the composition of pigments. The results demonstrated that the IDP isomerase is required for the physiological biosynthesis of terpenoids in plants. By these results the DXP pathway has become a bit more distinct, and the importance of the IDP isomerase for terpene biosythesis in higher plants was demonstrated. In addition, new chances for novel lead compounds for pharmaceutical application became evident. |