CRYSTAL STRUCTURE OF SERINE HYDROXYMETHYLTRANSFERASE

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 真核生物 三种氧化态中任何一种的一个碳单元 通过SHMT，有助于嘌呤，胸苷酸， 磷脂和甲硫氨酸。 SHMT是磷酸吡哆醛酶， 也需要四氢叶酸作为辅因子，后者携带 一个碳单位 不同的微分吸收光谱 中间体使得有可能表征一些步骤， 反应途径。 除了从一个碳单位的产生， 丝氨酸转化为甘氨酸，SHMT也被证明 催化大量的其他机械相关的反应， 不同的子阶段。 SHMT缺乏晶体结构， 阻碍了对其机制和功能作用的更全面的研究 酶中的基团。 这里建议的工作的目的是 测定了E. coli SHMT的X射线衍射分析 方法和使用该结构，其与辅酶的复合物， 底物和抑制剂，以及相关的突变体结构，以了解 这种酶的作用机制。 质粒表达的E.大肠杆菌SHMT 与5-甲酰四氢叶酸和甘氨酸络合， 其分辨率为2.8A，这些将用于解决 结构的重原子同晶置换法。 位点突变体 已经创建了这个SHMT，其他人可以通过 突变的作用的研究中的特定残基， 催化反应 在胸苷酸生物合成的循环中，SHMT是 这是循环中三种酶中唯一一种结构不 确定了 另外两个是二氢叶酸还原酶， 胸苷酸合成酶，这两者都是设计 化疗抗癌化合物。 相对知之甚少 其他两个代谢循环中的酶的结构， SHMT是活跃的。 SHMT对一个人的关键途径的中心地位 碳代谢使其成为一个富有成效的前景的设计，为其他 具有潜在治疗价值的化合物。 实现这一潜力 将需要一个知识的结构和理解其 机制，这是这里提出的工作的对象。

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.