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NMR Studies of DOXP Reductoisomerase and its Inhibitor Complex
Author(s) -
Englert Nadine E.,
Richter Christian,
Wiesner Jochen,
Hintz Martin,
Jomaa Hassan,
Schwalbe Harald
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000465
Subject(s) - chemistry , stereochemistry , active site , heteronuclear molecule , enzyme , nuclear magnetic resonance spectroscopy , biochemistry
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.