Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2

Abstract Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum . The recombinant production of the previously described Pf GDH1 as hexahistidyl-tagged proteins was optimized. Additionally, Pf GDH2 was cloned, recombinantly produced, and characterized. Like Pf GDH1, Pf GDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to Pf GDH1 but with slightly higher K m values for its substrates. The three-dimensional structure of hexameric Pf GDH2 was solved to 3.1 A resolution. The overall structure shows high similarity with Pf GDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in Pf GDH2 the subunit–subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in Pf GDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on Pf GDH2 than on Pf GDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, Pf GDH1 and Pf GDH3 are cytosolic proteins whereas Pf GDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum , which represent potential targets for the development of novel antimalarial drugs.