CYTIDYLYTRANSFERASE STRUCTURE & FUNCTION

磷脂酰转移酶（CCT）是生物合成磷脂酰胆碱的限速调控酶。磷脂酰胆碱是真核生物的主要磷脂。这个实验室的长期目标是了解这种酶的活性是如何被调节的。CCT属于胞基酰基转移酶家族，包括磷酸乙醇胺胞基转移酶（ECT）和甘油-3-磷酸胞基转移酶（GCT）。ECT参与真核生物磷脂酰乙醇胺合成的cdp -乙醇胺途径。GCT催化合成cd -甘油，在某些革兰氏阳性菌中合成磷壁酸的中间体。GCT是三种酶中最小的，与CCT和ECT的催化结构域有30- 35%的相似性。CCT是最复杂的，除了其催化结构域外，还包含几个调控区段。ECT包含两个类似GCT的序列拷贝，因此可能有两个催化结构域。用x射线晶体学测定了GCT的三维结构。本申请为进一步研究胞基转移酶家族的所有三个成员的结构和功能提供了基础。对CCT的研究将侧重于酶的催化片段的分析，并研究催化是如何被调控片段，特别是膜结合结构域调节的。研究将包括通过色氨酸荧光测量底物结合，定点诱变和结晶。GCT将从金属结合、底物结合和单体相互作用方面进行分析。ECT的两个结构域将通过定点诱变和单独表达来研究，以确定它们的功能。这些研究将深入了解这些胞基转移酶的催化机制，并探讨真核酶与其他酶段/结构域的相互作用如何调节催化作用。

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.