Cloning and characterization of a lipid-activated CTP: phosphocholine cytidylyltransferase from Caenorhabditis elegans: identification of a 21-residue segment critical for lipid activation

The genome of the nematode Caenorhabditis elegans contains several genes th at appear to encode proteins similar to CTP:phosphocholine cytidylyltransfe rase (CCT). We have isolated a 1044-nucleotide cDNA clone from a C elegans cDNA library that encodes the 347-amino acid version of CCT that is most si milar to previously-identified CCTs. Native and His-tagged forms were expre ssed and purified using a baculovirus expression system. The enzyme was max imally activated by 5 muM phosphatidylcholine:oleate (50:50) vesicles with a k(cat) value in the presence of lipid 37-fold greater than the k(cat) val ue in the absence of lipid. To localize the region of C elegans CCT critica l for lipid activation, a series of C-terminal truncation mutants was analy zed. CCT truncated after amino acids 225 or 245 was quite active in the abs ence of lipids and not further activated in the presence of lipids, support ing the concept that the lipid-activation segment is inhibitory to catalysi s in the absence of lipids. CCT truncated after amino acids 266, 281, or 31 9 was activated by lipid similar to wild-type enzyme. Kinetic analysis in t he absence of lipid revealed the lipid-independent CCT truncated after amin o acid 245 to have a k(cat) value 15-fold greater than either full-length C CT or CCT truncated after amino acid 266. We conclude that elements critica l for activation of C elegans CCT by lipids are contained within amino acid s 246-266, that this region is inhibitory in the absence of lipids, and tha t the inhibition is relieved by the association of the enzyme with lipid. ( C) 2001 Elsevier Science B.V. All rights reserved.