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 that appear to encode proteins similar to CTP:phosphocholine cytidylyltransferase (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 similar to previously-identified CCTs. Native and His-tagged forms were expressed and purified using a baculovirus expression system. The enzyme was maximally activated by 5 microM phosphatidylcholine:oleate (50:50) vesicles with a k(cat) value in the presence of lipid 37-fold greater than the k(cat) value in the absence of lipid. To localize the region of C. elegans CCT critical for lipid activation, a series of C-terminal truncation mutants was analyzed. CCT truncated after amino acids 225 or 245 was quite active in the absence of lipids and not further activated in the presence of lipids, supporting the concept that the lipid-activation segment is inhibitory to catalysis in the absence of lipids. CCT truncated after amino acids 266, 281, or 319 was activated by lipid similar to wild-type enzyme. Kinetic analysis in the absence of lipid revealed the lipid-independent CCT truncated after amino acid 245 to have a k(cat) value 15-fold greater than either full-length CCT or CCT truncated after amino acid 266. We conclude that elements critical for activation of C. elegans CCT by lipids are contained within amino acids 246-266, that this region is inhibitory in the absence of lipids, and that the inhibition is relieved by the association of the enzyme with lipid.
Friesen
J A
JA
Department of Biological Chemistry, 4417 Medical Science I, University of Michigan Medical Center, Ann Arbor, MI 48109-0606, USA.
Liu
M F
MF
Kent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019301:Oleic Acid / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000494:pharmacology; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
D018076:DNA, Complementary; D004075:Diglycerides; D007527:Isoenzymes; D008055:Lipids; D010712:Phosphatidic Acids; D019301:Oleic Acid; D019916:Choline-Phosphate Cytidylyltransferase; C013965:diolein