Crystallization and preliminary X-ray diffraction of malate dehydrogenase fromPlasmodium falciparum

Abstract

The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (Pf MDH) are reported. In order to gain a deeper understanding of the function and role of Pf MDH, the protein was purified to homogeneity. The purified protein crystallized in space group P1, with unit-cell parameters a = 72, b = 157, c = 159 Å , = 105, = 101, = 95. The resulting crystals diffracted to a maximal resolution of 2.24 Å and the structure has been solved by molecular replacement, with 16 monomers in the asymmetric unit. The 16 monomers are arranged into four independent tetramers, in agreement with previous reports demonstrating the tetrameric solution state of PfMDH. The X-ray structure of PfMDH is expected to clarify the differences in catalysis by PfMDH compared with other MDH family members and to provide a basis for the structure-based design of specific Pf MDH inhibitors as well as general MDH inhibitors.

Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of Plasmodium falciparum in vitro

“…Wild-type malate dehydrogenase ( EC 1.1.1.37 ) from Plasmodium falciparum ( Pf MDH-WT) was cloned, recombinantly expressed, purified and crystallised, as previously described [ 23 ]. Pf MDH-WT crystals belonged to P1 space group and diffracted to 2.4 Å. Molecular replacement yielded a clear solution of 4 tetramers in the asymmetric unit with R/R free of 0,25/0,26, respectively ( Table 1 ).…”

“…The final model has been deposited in PDB under accession code 5NFR . Pf MDH is a globular tetrameric protein [ 20 , 23 ], where each monomer is comprised of 326 residues, which form 9 alpha helixes and 11 beta -sheets ( Fig 1a and 1b ). Similarly to other NAD-dependent dehydrogenases, the active sites are located in the cleft between two domains: an N-terminal cofactor-binding domain containing a parallel structure of first six beta -sheets (Rossmann-fold) and C-terminal substrate-binding domain ( Fig 1a ) [ 21 , 24 ].…”

“…Values in parentheses correspond to the highest resolution shell. Data collection statistics are derived from previously reported article [ 23 ]…”

“…Cloning, recombinant expression and purification of Pf MDH were performed according to previously reported manuscript[ 23 ]. Site-directed mutagenesis was performed using sequence specific primers and pASK-Iba3- Pf MDH-WT plasmid as a template ( Table 4 ).…”

Identification and Validation of Novel Drug Targets for the Treatment of Plasmodium falciparum Malaria: New Insights

“…The mitochondrial MDH is absent in P. falciparum, being replaced by PfMQO (described in ETC section). The cytosolic MDH is present in P. falciparum (PfMDH) acting as a supplier of metabolites, such as malate, to the mitochondria and might be responsible for the generation of reducing equivalents to feed the respiratory chain [149].…”