Study of Glyoxalase II In Arabidopsis thaliana

Crowder和Makaroff作为更好地理解植物中的glycoprotein酶II在细胞生长和发育中的作用并获得glycoprotein酶II的结构/功能数据的研究的一部分，已经分离出对应于三个拟南芥glycoprotein酶II基因家族的五个cDNA，并正在对其进行表征。 本项目的目标是解决两个基本的和长期存在的问题，关于glycoprotease II：（1）glycoprotease II在植物中有什么功能？ （2）甘氨酸酶II的活性位点残基是什么？ 为了研究植物中谷胱甘肽酶II的功能，对A.将克隆、过表达和表征Thaliana。 具有纯化的酶将允许使用稳态动力学研究在体外进行底物特异性研究。第二部分是细胞化学和分子生物学研究，以确定植物生长和发育中glycoprotein酶II同工酶的作用。 为了解决在解毒和细胞增殖中的作用的glycoprotein酶II同工酶，分离和表征植物中的每一个glycoprotein酶II同工酶的基因中断的实验将进行。 为了确定谷胱甘肽酶II中的活性位点残基，将产生高度保守残基（包括推定的Zn结合配体）的突变，并且将表征所得突变酶的催化活性和金属结合。在glycoproteinase II蛋白质和它们的作用/性能的锌结合位点的表征，使用金属-b-内酰胺酶作为一个潜在的模型，将提供深入了解glycoproteinase II蛋白质的结构/功能关系，并确定用于催化的结构基序。谷胱甘肽酶系统由两种酶组成，谷胱甘肽酶I和谷胱甘肽酶II。 虽然在迄今为止研究的所有生物中都发现了谷胱甘肽酶的活性，但其在细胞中的确切作用仍然没有明确定义。在植物中，似乎谷胱甘肽酶系统在细胞解毒过程中起关键作用，并且还可能参与细胞增殖的调节。 本计画将探讨甘胺酸酶II的生理功能，并提供活性部位的结构资讯。

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.