Expression and characterization of N-terminal truncation of Plasmodium falciparum orotate phosphoribosyl transferrase enzyme

Abstract

Piasmodium falciparum, the causative agent of the most lethal form of human malaria, totally depends on de novo pyrimidine biosynthetic pathway. A gene encoding orotate phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate (PP[subscript i]) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified from P. falciparum (pfOPRT), and was exceptional in that it contained an amino-terminal extension of 66 amino acids, making the longest amino acid sequence (218 amino acids). This unique sequence remains uncharacterized the importance in the parasite. In this study, the cDNA of the full-length and N-terminal truncated pfOPRT gene was cloned and expressed. The both types of the recombinant pfOPRT were purified from the E. coli lysate by nickel metal-affinity chromatography. After that The both types of the recombinant pfOPRT were analyzed by SDS-PAGE and Western blot. Furthermore, the studying in activity and stability were included. The results revealed that the full-length and N-terminal truncated pfOPRT had a molecular mass of 35 kDa and 30 kDa, respectively. The specific activity of the N-terminal truncated pfOPRT was decreased about 10-fold when compared with the full-length pfOPRT. The stability of the N-terminal truncated pfOPRT was lower than the full-length pfOPRT. So, it concluded that the N-terminal truncated pfOPRT, which had a molecular weight of 30 kDa, had less specific activity and stability than the full-length pfOPRT. These evidences provided that the N-terminal extension of pfOPRT may have an important role in the activity and stability of this enzyme, therefore it may be the new target for structure-based antimalarial drug design. To block this extension part will take the action on parasite survival by limiting the pyrimidine base biosynthesis.