Identification, characterization, and cellular localization of Leishmania major CTP:phosphocholine...

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis, the second largest parasitic killer in the world behind malaria. A large percentage of Leishmania membrane phospholipids is phosphatidylcholine (PC), formed via the Kennedy pathway, where the enzyme CTP: phosphocholine cytidylyltransferase (CCT) catalyzes the second, rate limiting step. Leishmania major CCT was expressed in non-pathogenic Leishmania tarentolae and exhibited activity that increased 10-fold in the presence of PC:oleate lipid vesicles. Confocal microscopy of L. tarentolae expressing L. major CCT fused to a red fluorescent protein revealed the enzyme is cytoplasmic but may associate with internal membranes. A truncated mutant of L. major CCT containing the catalytic domain was expressed in Escherichia coli and in vitro analysis of the enzyme showed catalysis was divalent cation-dependent and yielded a V_max of 374 nmol/min/mg and K_m values of 0.0648 mM and 3.74 mM, respectively, for the substrates CTP and phosphocholine.