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CRYSTAL STRUCTURE OF SERINE HYDROXYMETHYLTRANSFERASE

丝氨酸羟甲基转移酶的晶体结构

基本信息

批准号：
2415211
负责人：
HARLAN Tonie WRIGHT
金额：
$ 14.36万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 2000-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2415211
关键词：
X ray crystallography chemical models cofactor computer program /software computer simulation crystallization enzyme complex enzyme mechanism enzyme structure enzyme substrate enzyme substrate complex hydroxymethyltransferases isomorphous substitution protein purification pyridoxal phosphate site directed mutagenesis tetrahydrofolates

项目摘要

Serine hydroxymethyl transferase (SHMT) is a ubiquitous enzyme which lies at the center of one carbon metabolism in both prokaryotes and eukaryotes. One carbon units carried in any of three oxidation states by SHMT are contribute to the biosynthesis of purines, thymidylate, phospholipids, and methionine. SHMT is a pyridoxal phosphate enzyme and also requires tetrahydrofolate as a cofactor, the latter of which carries the one carbon units. The differential absorbance spectra of various intermediates has made it possible to characterize some of the steps of the reaction pathway. Beside the generation of one carbon units from the transformation of serine into glycine, SHMT has also been shown to catalyse a large number of other, mechanistically related reactions with different substages. The lack of a crystal structure of SHMT has hampered a fuller investigation of its mechanism and roles of functional groups in the enzyme. The aims of the work proposed here are to determine the crystal structure of E. coli SHMT by x-ray diffraction methods and to use this structure, its complexes with coenzymes, substrates and inhibitors, and related mutant structures to understand the mechanism of this enzyme. Crystals of plasmid-expressed E. coli SHMT complexed with 5-formyltetrahydrofolate and glycine have been obtained, which diffract to a resolution 2.8A and these will be used to solve the structure by the heavy atom isomorphous replacement method. Site mutants of this SHMT have already been created, and others can be made by mutagenesis for the study of the role of specific residues in the catalytic reaction. In the cycle for thymidylate biosynthesis, SHMT is the only one of the three enzymes of the cycle whose structure has not been determined. The other two are dihydrofolate reductase and thymidylate synthase, both of which have been targets for the design of chemotherapeutic anticancer compounds. Relatively little is known about the structures of the enzymes in the other two metabolic cycles in which SHMT is active. The centrality of SHMT to the critical pathway of one carbon metabolism makes it a fruitful prospect for the design of other compounds of potential therapeutic value. Realization of this potential will required a knowledge of its structure and an understanding of its mechanism, which are the objects of the work proposed here.

丝氨酸羟甲基转移酶(SHMT)是一种普遍存在的酶在原核生物和生物体内碳代谢的中心真核生物。一个碳单位，以三种氧化态中的任何一种形式携带通过SHMT促进了嘌呤、胸腺嘧啶、磷脂和蛋氨酸。SHMT是一种吡哆醛磷酸酶，还需要四氢叶酸作为辅因子，后者携带一个碳单位。几种不同化合物的差示吸收光谱中间体使得描述一些步骤成为可能反应途径。除了产生一个碳单位外，丝氨酸转化为甘氨酸，SHMT也被证明催化大量其他与机械有关的反应不同的子阶段。SHMT的晶体结构的缺乏阻碍了对其机制和功能作用的更充分的研究酶中的基团。这里提出的工作的目的是 X射线衍射法测定大肠杆菌SHMT的晶体结构方法并利用这种结构，它与辅酶的络合物，底物和抑制剂，以及相关的突变结构这种酶的作用机制。原核表达的大肠杆菌SHMT的晶体与5-甲酰基四氢叶酸和甘氨酸形成络合物，它绕射到2.8A分辨率，这些将被用来解决结构采用重原子同象取代法。位点突变体已经创建了这个SHMT，其他的可以由诱变技术用于研究特定残基在细菌感染中的作用催化反应。在胸苷生物合成的循环中，SHMT是循环中三种酶中唯一一种其结构没有已经确定了。另外两种是二氢叶酸还原酶和胸苷合成酶，这两个都是设计的目标化疗抗癌化合物。人们对此知之甚少另外两个代谢周期中的酶的结构 SHMT处于活动状态。SHMT对One关键路径的中心性碳代谢使其成为一种富有成效的设计方案具有潜在治疗价值的化合物。实现这一潜力遗嘱需要了解它的结构，并理解它的机制，这些都是这里提出的工作的目标。