As 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.
