CTP:phosphocholine cytidylyltransferase (CCT) is rate-limiting and regulatory for the biosynthesis of phosphatidylcholine, the major eukaryotic phospholipid. A long-term goal of this laboratory has been to understand how the activity of this enzyme is regulated. CCT belongs to a family of cytidylyltransferases which included CTP:phosphoethanolamine cytidylytransferase (ECT) and CTP:glycerol-3-phosphate cytidylyltransferase (GCT). ECT participates in the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis in eukaryotes. GCT catalyzes the synthesis of CDP-glycerol, an intermediate in the synthesis of teichoic acids in certain gram positive bacteria. GCT, the smallest of the three enzymes, shares 30-35 percent identity with catalytic domains of CCT and ECT. CCT is the most complex, containing several regulatory segments in addition to its catalytic domain. ECT contains two copies of sequences similar to GCT, and thus may have two catalytic domains. A three-dimensional structure of GCT has just been determined by x- ray crystallography. This application proposes further studies on the structure and function of all three members of this cytidylyltransferase family. Studies on CCT will emphasize analysis of the catalytic fragment of the enzyme, and investigate how catalysis is modulated by the regulatory segments, in particular, the membrane-binding domain. Studies will include measurement of substrate binding by intrinsic tryptophan fluorescence, site-directed mutagenesis, and crystallization. GCT will be analyzed with respect to metal-binding, substrate- binding, and interaction of monomers. The two domains of ECT will be investigated by site-directed mutagenesis and by separate expression to determine their functions. These studies will provide insight into the mechanism of catalysis by these cytidylyltransferases, and probe how catalysis is modulated by interactions with other segments/domains in the eukaryotic enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060510-03
Application #
6498723
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Ikeda, Richard A
Project Start
2000-02-01
Project End
2004-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
3
Fiscal Year
2002
Total Cost
$286,351
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Kent, Claudia; Gee, Patricia; Lee, Si Young et al. (2004) A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola. Mol Microbiol 51:471-81
Yuan, Chong; Kent, Claudia (2004) Identification of critical residues of choline kinase A2 from Caenorhabditis elegans. J Biol Chem 279:17801-9
Helmink, Beth Ann; Braker, Jay D; Kent, Claudia et al. (2003) Identification of lysine 122 and arginine 196 as important functional residues of rat CTP:phosphocholine cytidylyltransferase alpha. Biochemistry 42:5043-51
Pattridge, Katherine A; Weber, Christian H; Friesen, Jon A et al. (2003) Glycerol-3-phosphate cytidylyltransferase. Structural changes induced by binding of CDP-glycerol and the role of lysine residues in catalysis. J Biol Chem 278:51863-71
Gee, Patricia; Kent, Claudia (2003) Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization. Biochim Biophys Acta 1648:33-42
Sanker, S; Campbell, H A; Kent, C (2001) Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP:glycerol-3-phosphate cytidylyltransferase. J Biol Chem 276:37922-8
Friesen, J A; Park, Y S; Kent, C (2001) Purification and kinetic characterization of CTP:phosphocholine cytidylyltransferase from Saccharomyces cerevisiae. Protein Expr Purif 21:141-8
Friesen, J A; Liu, M F; Kent, C (2001) Cloning and characterization of a lipid-activated CTP:phosphocholine cytidylyltransferase from Caenorhabditis elegans: identification of a 21-residue segment critical for lipid activation. Biochim Biophys Acta 1533:86-98
Campbell, H A; Kent, C (2001) The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization. Biochim Biophys Acta 1534:85-95