Crystallization and preliminary X-ray diffraction studies of monomeric isocitrate dehydrogenase by the MAD method using Mn atoms

NADP(+) -dependent isocitrate dehydrogenase (E.C. 1.1.1.42; IDH) is an enzy me of the Krebs cycle and catalyzes the oxidative decarboxylation reaction from dl-isocitrate to alpha -ketoglutarate, with a concomitant reduction of the coenzyme NADP(+) to NADPH. Single crystals of monomeric IDH from Azoto bacter vinelandii in complex with DL-isocitrate and Mn2+ were obtained by t he hanging-drop vapour-diffusion method at room temperature. One crystal di ffracted to a resolution of 2.9 Angstrom and was found to belong to the ort horhombic system; the space group was determined to be P2(1)2(1)2(1), with unit-cell parameters a = 108.4, b = 121.7, c = 129.7 Angstrom. The asymmetr ic unit contains two molecules of monomeric IDH, corresponding to a VM valu e of 2.66 Angstrom (3) Da(-1). The crystals were frozen in a capillary by a flash-cooling technique and MAD data were collected using Mn atoms as anom alous scatterers on beamline BL41XU at SPring-8, Japan. The positions of tw o Mn atoms binding to two independent IDH molecules were located from Bijvo et difference Patterson maps.