Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile

• Published in: Molecular and biochemical parasitology Vol. 134; no. 2; pp. 245 - 255
• Main Authors: Krungkrai, Sudaratana R, Aoki, Sayaka, Palacpac, Nirianne Marie Q, Sato, Dan, Mitamura, Toshihide, Krungkrai, Jerapan, Horii, Toshihiro
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2004
• Subjects: Amino Acid Sequence; Animals; Antimalarials - pharmacology; Binding Sites - genetics; Catalytic Domain - genetics; Chromatography; Cloning, Molecular; Dimerization; DNA, Protozoan - chemistry; DNA, Protozoan - isolation & purification; Enzyme Inhibitors - pharmacology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genes, Protozoan; Kinetics; Molecular Sequence Data; Molecular Weight; Orotate phosphoribosyltransferase; Orotate Phosphoribosyltransferase - antagonists & inhibitors; Orotate Phosphoribosyltransferase - chemistry; Orotate Phosphoribosyltransferase - genetics; Orotate Phosphoribosyltransferase - metabolism; Plasmodium falciparum; Plasmodium falciparum - enzymology; Plasmodium falciparum - genetics; Protein Subunits; Pyrimidine metabolism; pyrimidines; Recombinant expression; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Salmonella typhimurium - genetics; Sequence Alignment; Substrate Specificity; FPLC; Recombinant expression; DTT; OPRT; PRPP; OMPDC; PP i; Ni 2+-NTA; Orotate phosphoribosyltransferase; Pyrimidine metabolism; Plasmodium falciparum; OMP; PMSF; RT-PCR; IPTG; pfOPRT; UMP; HPLC
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2003.12.006

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, relies on de novo pyrimidine biosynthesis. A gene encoding orotate phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway catalyzing formation of orotidine 5′-monophosphate (OMP) and pyrophosphate (PP i) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified from P. falciparum ( pfOPRT). The deduced amino acid sequence for pfOPRT was compared with OPRTs from other organisms and found to be most similar to that of Escherichia coli. The catalytic residues and consensus sequences for substrate binding in the enzyme were conserved among other organisms. The pfOPRT was exceptional in that it contained a unique insertion of 20 amino acids and an amino-terminal extension of 66 amino acids, making the longest amino acid sequence (281 amino acids with a predicted molecular mass of 33 kDa). The cDNA of the pfOPRT gene was cloned, sequenced and functionally expressed in soluble form. The recombinant pfOPRT was purified from the E. coli lysate by two steps, nickel metal-affinity and gel-filtration chromatography. From 1 l E. coli culture, 1.2–1.5 mg of pure pfOPRT was obtained. SDS–PAGE revealed that the pfOPRT had a molecular mass of 33 kDa and analytical gel-filtration chromatography showed that the enzyme activity eluted at approximately 67 kDa. Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it was confirmed that the native enzyme exists in a dimeric form. The steady state kinetics of initial velocity and product inhibition studies indicate that the enzyme pfOPRT follows a random sequential kinetic mechanism. Compounds aimed at the pfOPRT nexus may act against the parasite through at least two mechanisms: by directly inhibiting the enzyme activity, or be processed to an inhibitor of thymidylate synthase. This study provides a working system with which to investigate new antimalarial agents targeted against P. falciparum OPRT.