STRUCTURE/FUNCTION OF PYRUVOYL AND PLP DEPENDENT ENZYMES

本研究的目的是了解结构-功能 氨基酸脱羧酶的相互关系和作用机制 磷酸吡哆醛（PLP）依赖性酶的主要类别， 可获得的结构信息相对较少。 脱羧酶是 重要的治疗靶点，产生生物胺，如 组胺多巴胺和多胺 结构信息 脱羧酶及其抑制剂复合物是理解 不同的PLP酶足够详细，以帮助设计药物 针对其特定的酶活性。 一个PLP-1的X-射线结构、基因序列和效应特性， 依赖性鸟氨酸脱羧酶（ODC）。 我们提出 使用X射线晶体学，辅以现场技术， 定向诱变和稳态动力学，检查的作用， 参与作用机制和效应物激活的关键残基 L.30a的ODC。 抑制剂复合物的结构将回答 关于“封闭”和“开放”形式的ODC的性质和 催化中间体的立体化学 X射线结构和动力学 定点突变体的性质将使特定的分配 对负责底物特异性的氨基酸残基的作用， 结合、催化机制、GTP效应子作用和亚基 交互. 基于来自L.30a的ODC结构的序列比较使我们得出： 这表明，至少有两个不同的结构家族， 脱羧酶 X射线质量的晶体已经生产了两个 第二类脱羧酶的例子，小鼠ODC（其非常 与人类和锥虫ODC密切相关）和生物合成 精氨酸脱羧酶（bADC）。杆菌 它们的X射线结构 酶将被确定和机制研究类似于那些 L.30a为ODC提出的建议将扩展到这些系统。 小鼠ODC-DFMO（二氟甲基鸟氨酸）晶体，一种自杀抑制剂 用于治疗非洲昏睡病的ODC，也被 得到了 这些酶代表了一类新的高度调节的， 用于X射线结构分析的治疗靶点。 他们还分享了 由抗酶结合引起的失活的新模型和条件 以产生脱羧酶-抗酶复合物晶体。

The goal of this research is to understand the structure-function relationships and mechanisms of action of amino acid decarboxylases, a major class of pyridoxal phosphate (PLP)-dependent enzymes for which relatively little structural information is available. Decarboxylases are important therapeutic targets, generating biogenic amines such as histamine, dopamine, and polyamines. Structural information on decarboxylases and their inhibitor complexes are necessary to understand different PLP enzymes in sufficient detail to aid in the design of drugs targeted against their specific enzymatic activities. The X-ray structure, gene sequence and effector properties of a PLP- dependent ornithine decarboxylase (ODC) have been determined. We propose to use X-ray crystallography, supplemented with the techniques of site- directed mutagenesis and steady state kinetics, to examine the roles of key residues involved in the mechanism of action and effector activation of ODC from L.30a. Structures of inhibitor complexes will answer questions about "closed" and "open" forms of ODC and the nature and stereochemistry of catalytic intermediates. X-ray structures and kinetic properties of site-directed mutants will enable the assignment of specific roles to amino acid residues responsible for substrate specificity and binding, catalytic mechanism, GTP effector action, and subunit interactions. Sequence comparisons based on the structure of ODC from L.30a led us to suggest that there are at least two distinct structural families of decarboxylases. X-ray quality crystals have been produced for two examples of second class of decarboxylases, mouse ODC (which is very closely related to the human and trypanosomal ODCs) and biosynthetic arginine decarboxylase (bADC) from E. coli. The X-ray structures of these enzymes will be determined and mechanistic studies similar to those proposed for the ODC from L.30a will be extended to these systems. Crystals of mouse ODC-DFMO (difluoromethyl ornithine), a suicide inhibitor of ODC used in the treatment of African sleeping sickness, have also been obtained. These enzymes represent a new class of highly regulated, therapeutic targets for X-ray structural analysis. They also share a novel model of inactivation resulting from antizyme binding and conditions to produce decarboxylase-antizyme complex crystals will be screened.