CYTIDYLYTRANSFERASE STRUCTURE & FUNCTION

CTP：磷酸胆碱胞苷酰转移酶（CCT）对磷脂酰胆碱（主要的真核磷脂）的生物合成具有限速和调节作用。 该实验室的长期目标是了解这种酶的活性是如何调节的。 CCT属于胞苷酰转移酶家族，其包括CTP：磷酸乙醇胺胞苷酰转移酶（ECT）和CTP：甘油-3-磷酸胞苷酰转移酶（GCT）。 ECT参与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CDP-甘油的合成，CDP-甘油是某些革兰氏阳性菌中磷壁酸合成的中间体。GCT是三种酶中最小的一种，与CCT和ECT的催化结构域具有30- 35%的同一性。 CCT是最复杂的，除了其催化结构域之外，还包含几个调节片段。 ECT含有与GCT相似的序列的两个拷贝，因此可能具有两个催化结构域。 刚用X射线晶体学测定了GCT的三维结构。 本申请提出了对该胞苷酰转移酶家族的所有三个成员的结构和功能的进一步研究。 对CCT的研究将强调对酶催化片段的分析，并研究催化如何被调节片段，特别是膜结合结构域调节。 研究将包括通过内源性色氨酸荧光、定点诱变和结晶测量底物结合。将分析GCT的金属结合、底物结合和单体相互作用。 ECT的两个结构域将通过定点诱变和通过单独表达来研究以确定它们的功能。 这些研究将提供深入了解这些胞苷酰转移酶的催化机制，并探讨如何通过与真核酶中的其他片段/结构域的相互作用来调节催化。

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.