Cloning and Expression of Malarial Pyrimidine Enzymes

Abstract

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.

Genetic crosses and complementation reveal essential functions for thePlasmodiumstage-specific actin2 in sporogonic development

“…The antiserum recognizing actin2 was produced in mice immunized with protein purified from Escherichia coli. The complete ORF of actin2 was cloned in plasmid pET23d and expression was done in E. coli BL21(DE3)pLysS containing the plasmid pMICO (Christopherson et al, 2004). The protein was isolated from insoluble inclusion bodies.…”

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

“…The three enzymes, carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydroorotase from Plasmodium falciparum were cloned and expressed and shown to contain unusual protein inserts when compared with sequences of these enzymes from other organisms (Christopherson et al, 2004). Aspartate transcarbamylase (Mejias-Torres and Zimmermann, 2002) and dihydroorotase (Robles Lopez et at., 2006) from Toxoplasma gondii were cloned, purified and characterized.…”

“…The genes encoding the two enzymes in P. falciparum are distinct and located on different chromosomes. The two enzymes are produced separately, but they get cross-linked later and appear as a multienzyme complex with different kinetic properties from the host bifunctional protein complex (Christopherson et al, 2004; Krungkrai et al, 2004 and 2005; Krungkrai and Krungkrai, 2016). Proteomic data and structural modeling showed that an insertion of a low complexity amino acid sequence is responsible for this interaction of orotate phosphoribosyltransferase and OMP decarboxylase in P. falciparum (Imprasittichail et al, 2014).…”

Targeting Purine and Pyrimidine Metabolism in Human Apicomplexan Parasites

“…The other important enzymes in de novo UMP synthesis comprise aspartate carbamoyltransferase (ATCase; EC 2.1.3.2), which catalyses the second step, and whose gene and product have been characterised in T. gondii [37], dihydroorotase (DHOase; EC 3.5.2.3), which catalyses the third step, whose gene has been cloned from P. falciparum [38] and T. gondii [39], dihydroorotate dehydrogenase (DHODH; EC 1.3.3.1) the fourth (see later), orotate phosphoribosyltransferase (OPRT; EC 2.4.2.10) the fifth, and orotidine 5′-monophosphate decarboxylase (OMPDC; EC 4.1.1.23), the sixth, Fig. ( 1 ).…”

“…( 1 ). In P. falciparum and T. gondii , separate genes encode OPRT and OMPDC [38], which have been shown in the malaria parasite to associate into a complex comprising two subunits of each enzyme [40, 41], with different kinetic properties from the host UMP synthase, where these activities are combined on a single bifunctional protein. Analogues of orotate (the substrate of OPRT) substituted at the 5′ position have been shown to inhibit pyrimidine synthesis in P. falciparum and in the rodent parasites P. berghei [42, 43] and P. yoelii [44].…”