NMR studies of DOXP reductoisomerase and its inhibitor complexes

1-deoxy-D-xylulose 5-phosphat reductoisomerase (DOXP reductoisomerase, DXR, EC1.1.1.267) catalyses the second step of the 2-C-methyl-Derythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis, which is used by most bacteria, apicomplexan parasites and the plastids of plants. The protein is absent in 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 optimization and partial backbone assignment and secondary structure prediction of E. coli DXR by heteronuclear NMR analysis for further NMR-aided drug discovery. Perdeuterated 15N, 13C labelled samples were prepared under oxygen exclusion in presence of Mg2+, NADPH and the inhibitor FR-900098, which represents a close derivative of fosmidomycin. 1H and 15N backbone assignment was achieved for 44% of the primary structure, and 13C 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.