The investigation of plant dipeptidyl peptidase III (DPP III) lead to an unexpected discovery, the existence of an enzyme with dual function (Nudix and DPP III), which also shows the atypic Nudix hydrolase activity against the isopentenyl diphosphate (IPP) substrate. For the purpose of structural characterization of the Nudix domain of this interesting enzyme, and based on the significant similarity of primary structures, Nudix hydrolase from amoeba Dictyostelium discoideum (DdNudix) was selected for this study. Nudix hydrolases are members of superfamily of hydrolytic Mg2+-dependent enzymes acting as phosphatases or pyrophosphohydrates. The wild type and inactive E82A mutant DdNudix, labeled with histidine tags, were over-expressed in Escherichia coli bacteria and purified by affinity chromatography and gel filtration. The conditions for crystallization of DdNudix protein were optimized. Using anomalous scattering, the structure of the selenomethionine labeled wild-type protein was resolved up to the resolution of 2.4 Å. Structure of the protein mutant, E82A, in complex with IPP substrate was solved with the molecular replacement method up to the resolution of 1.6 Å. Detailed structural analysis revealed similarities and differences of Nudix hydrolases as well as isopentenyl diphosphate isomerases.