CYTIDYLYTRANSFERASE STRUCTURE & FUNCTION

CTP：磷胆碱胞苷转移酶(CCT)是磷脂酰胆碱生物合成的限速和调节酶，磷脂酰胆碱是真核生物中主要的磷脂。这个实验室的一个长期目标是了解这种酶的活性是如何调节的。CCT属于胞苷转移酶家族，包括CTP：磷酸乙醇胺胞苷转移酶(ECT)和CTP：甘油-3-磷酸胞苷转移酶(GCT)。ECT参与了真核生物中磷脂酰乙醇胺生物合成的CDP-乙醇胺途径。GCT催化合成CDP-甘油，CDP-甘油是某些革兰氏阳性细菌合成磷壁酸的中间产物。GCT是三种酶中最小的一种，与CCT和ECT的催化域有30%-35%的同源性。CCT是最复杂的，除了它的催化结构域之外，还包含几个调节片段。ECT含有与GCT相似的两个拷贝的序列，因此可能有两个催化结构域。用X射线结晶学测定了GCT的三维结构。这项申请提出了对该胞苷酰转移酶家族所有三个成员的结构和功能的进一步研究。对CCT的研究将侧重于对酶的催化片段的分析，并调查调控片段，特别是膜结合结构域是如何调节催化的。研究将包括通过固有的色氨酸荧光测量底物结合、定点突变和结晶。GCT将从金属结合、底物结合和单体的相互作用方面进行分析。ECT的这两个结构域将通过定点突变和分离表达来研究，以确定它们的功能。这些研究将深入了解这些胞苷转移酶的催化机制，并探索催化是如何通过与真核酶中其他片段/结构域的相互作用来调节的。

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.