The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
Abstract:The natural formation of the bioactive C 17 -polyacetylenes (−)-(R)-panaxynol and panaxydol was analyzed by 13 C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13 CO 2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-
13C 6 ]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13 C-labeled isotopologues and one single labeled isotopologue were observed in the C 17 -polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13 C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13 CO 2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots.
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