Crystallization and preliminary X-ray diffraction analysis of malic enzyme from pigeon liver

Abstract

Recombinant pigeon-liver malic enzyme was expressed in Escherichia coli and puri®ed to homogeneity. Two different crystal forms were grown by the hanging-drop vapour-diffusion method. Both types of crystals belong to the tetragonal space group P4 2 22, with unit-cell dimensions a = b = 163.8, c = 174.3 A Ê for the octahedral crystals and a = b = 124.5, c = 179.2 A Ê for the rod-like crystals. X-ray diffraction data were collected at 100 K using a synchrotron-radiation X-ray source. The Matthews parameter suggests that there are four and two molecules per asymmetric unit for the larger and the smaller tetragonal unit cells, respectively.

Structural studies of the pigeon cytosolic NADP+‐dependent malic enzyme

“…Crystals of pigeon c‐NADP‐ME were first reported more than 30 years ago (Hsu and Lardy 1967), although those crystals were not characterized by X‐ray diffraction studies. Recently, we reported two new crystal forms of this enzyme, grown in the presence of d ‐malate as a substrate‐analog inhibitor (Tsai et al 1999). Although one of these crystal forms only diffracted X‐rays to 4‐Å resolution, a data set to 2.9‐Å resolution was collected for the other crystal form (Tsai et al 1999).…”

“…Recently, we reported two new crystal forms of this enzyme, grown in the presence of d ‐malate as a substrate‐analog inhibitor (Tsai et al 1999). Although one of these crystal forms only diffracted X‐rays to 4‐Å resolution, a data set to 2.9‐Å resolution was collected for the other crystal form (Tsai et al 1999). The structure solution by the molecular replacement method was able to locate only one monomer of the enzyme in the asymmetric unit.…”

“…The tetramer of the enzyme can be generated by the crystallographic symmetry from the monomer, and there are packing contacts among the tetramers in the crystal. Crystal structure refinement based on this molecular replacement solution was not successful, however, due to the poor quality of the diffraction data (Tsai et al 1999).…”

Dynamic Description of the Catalytic Cycle of Malate Enzyme: Stereoselective Recognition of Substrate, Chemical Reaction, and Ligand Release

“…15 We failed to get the crystal form of the DMA−ME complex in Protein Data Bank. 21,59 DMA−ME binding conformation was based on docking of the substrate to enzyme. In Figure 3B, two oxygen atoms (O3 and O4) pointed toward the catalytic Mn 2+ ion and stayed at distances of 2.10 and 2.14 Å, respectively.…”