Study of Glyoxalase II In Arabidopsis thaliana

克劳德和马卡洛夫作为一项研究的一部分，以更好地了解乙草酸酶II在植物细胞生长发育中的作用(S)并获得乙草酸酶II的结构/功能数据，已经分离到与三个拟南芥乙二醛酶II基因家族相对应的五个基因。这个项目的目标是解决关于乙草酸酶II的两个基本和长期存在的问题：(1)乙草酸酶II在植物中有什么功能(S)？和(2)乙二醛酶II中的活性中心残基是什么？为了确定乙醛酸酶II在植物中的功能(S)，我们将克隆、高效表达和鉴定拟南芥乙醛酸酶II的每一种同工酶。拥有纯化的酶将允许使用稳态动力学研究在体外进行底物专一性研究。该项目的第二个方面涉及细胞化学和分子研究，以确定乙二醇酶II同工酶在植物生长和发育中的作用(S)。为了解决乙二酸酶II同工酶在解毒和细胞增殖中的作用，将进行实验，以分离和鉴定在每种乙草酸酶II同工酶中含有基因中断的植物。为了确定乙醛酸酶II的活性中心残基，将产生高度保守的残基突变(包括可能的锌结合配体)，并对所产生的突变酶的催化活性和金属结合进行表征。利用金属β-内酰胺酶作为一个潜在的模型，表征乙醛酶II蛋白中的锌结合部位及其作用/性质，将有助于深入了解乙醛酶II蛋白的结构/功能关系，并确定用于催化的结构基序。乙草酸酶系统由乙草酸酶I和乙醛酸酶II两种酶组成。虽然到目前为止在所有研究的生物中都发现了乙草酸酶的活性，但它在细胞中的确切作用仍然没有明确的定义。在植物中，乙醛酶系统似乎在细胞解毒过程中起着关键作用，也可能参与细胞增殖的调节。本项目将研究乙二酸酶II的生理功能，并提供有关活性部位的结构信息。

Crowder and MakaroffAs part of a study to better understand the role(s) of glyoxalase II in cellular growth and development in plants and obtain structure/function data on glyoxalase II, five cDNA's corresponding to three Arabidopsis glyoxalase II gene families have been isolated and are being characterized. The goal of this project is to address two fundamental and long standing questions concerning glyoxalase II: (1) What function(s) does glyoxalase II have in plants? and (2) What are the active site residues in glyoxalase II? In order to determine the function(s) of glyoxalase II in plants, each of the glyoxalase II isozymes from A. thaliana will be cloned, overexpressed, and characterized. Having the purified enzymes will allow for substrate specificity studies to be performed in vitro using steady-state kinetic studies. The second aspect of this part of the project involves cytochemical and molecular studies to determine the role(s) of glyoxalase II isozymes in plant growth and development. To address the role of glyoxalase II isozymes in detoxification and cell proliferation, experiments to isolate and characterize plants containing gene disruptions in each of the glyoxalase II isozymes will be conducted. In order to determine the active site residues in glyoxalase II, mutations of highly conserved residues (including the putative Zn binding ligands) will be generated, and the resulting mutant enzymes will be characterized for catalytic activity and metal binding. Characterization of the Zn binding sites in glyoxalase II proteins and their role/properties, using the metallo-b-lactamases as a potential model, will provide insight into structure/function relationships in glyoxalase II proteins and identify the structural motif used for catalysis. The glyoxalase system consists of two enzymes, glyoxalase I and glyoxalase II. While glyoxalase activity is found in all organisms studied to date, its exact role in the cell has still not been clearly defined. In plants it appears that the glyoxalase system plays a critical role in cellular detoxification processes and may also be involved in the regulation of cell proliferation. This project will address the physiological function of glyoxalase II and provide structural information about the active sites.