Molecular cloning of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum.
CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulatory enzyme in the synthesis of phosphatidylcholine, the major membrane phospholipid, in Plasmodium. The structural gene encoding CCT was isolated from the human malaria parasite Plasmodium falciparum. This was achieved using the PCR to amplify genomic DNA with degenerate primers constructed on the basis of conserved regions identified within yeast and rat liver CCT molecules, and using the PCR product to screen a genomic library. The P. falciparum CCT gene encodes a protein of 370 amino acids (42. 6 kDa) and displays 41-43% similarity (28-29% identity) to CCT molecules of the other organisms cloned to date. The central domain of CCT, proposed as the catalytic domain of the CTP-transfer reaction, shows 68-72% similarity and 48-55% identity among P. falciparum, human, rat and yeast enzymes. This gene is present in a single copy, as determined by Southern-blotting of genomic DNA, and located on chromosome 13 of P. falciparum. Large transcripts were detected by Northern analysis and indicate that this gene is expressed in the asexual intraerythrocytic stages. The coding region of the P. falciparum CCT gene was inserted into an Escherichia coli expression vector to confirm the function of the CCT product. The recombinant CCT expressed in E. coli is catalytically active, as evidenced by the conversion of phosphocholine to CDP-choline.