CYTIDYLYTRANSFERASE STRUCTURE & FUNCTION

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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.