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摘要
1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase (EC1.1.1.267) catalyses the second step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. The enzyme is used by most bacteria, apicomplexan parasites and the plastids of plants, but not by humans, and therefore represents an attractive target for antibacterial, antiparasitic and herbicidal compounds. Fosmidomycin, an inhibitor of DXR, has been found to be active against bacterial infections and malaria in early clinical studies. Here, we report sample optimisation, partial backbone assignment and secondary-structure prediction of E. coli DXR by heteronuclear NMR analysis for further NMR-aided drug discovery. Perdeuterated (15)N,(13)C-labelled samples were prepared under oxygen exclusion in the presence of Mg(2+) , NADPH and the inhibitor FR-900098, a close derivative of fosmidomycin. (1)H and (15)N backbone assignment was achieved for 44 % of the primary structure, and (13)C backbone assignment was achieved for 50 % of the primary structure. Comparison with previously solved crystal structures revealed that the assigned fragments were located mainly in helical regions on the solvent-exposed surface of the enzyme. Torsion angle likelihood obtained from shift and sequence similarity (TALOS) was used for secondary structure prediction, resulting in agreement with eight available crystal structures; deviations could be observed for the catalytic loop region.
- 出版日期2011-2-11
