Molecular characterization of glyoxalase I from Arabidopsis thaliana

All biological cells produce methylglyoxal, a toxic 2-oxoaldehyde, as a by-product of primary metabolic processes such as carbohydrate oxidation. Methylglyoxal can cause covalent modification of DNA, RNA and proteins and has been implicated in various human disease states such as diabetes, Alzheimer's disease, acute anxiety disorder and cancer. The consequences of methylglyoxal accumulation in other organisms are less clear, but all eukaryotes and bacteria seem to possess a pathway for its detoxification based on the glyoxalase system. This consists of two enzymes that convert cytotoxic 2-oxoaldehydes to non-toxic 2-hydroxycarboxylicacids with the aid of the abundant intracellular thiol, glutathione. The first enzyme in this pathway, glyoxalase I (GLXI), is of special interest because two different forms have been reported to date: many organisms possess a zinc-dependent form, but it was recently discovered that other organisms possess a nickel-dependent form ofGLXl. Relatively little is known about the glyoxalase system in plants, so these enzymes were investigated in Arabidopsis thaliana, a species whose complete genome 'has been sequenced. Bioinformatic analysis suggested that this plant contains three distinct genes encoding GLX1, whereas most organisms possess only a single GLX1 gene. Experiments in which the plant proteins were functionally expressed in yeast and bacterial GUO mutants demonstrated that these three proteins all possess GLXI activity in vivo. Recombinant proteins were also expressed to study their enzymatic properties in vitro. One isoform was found to be a zinc-dependent enzyme, whereas the other two isoforms showed an apparent preference for nickel. This is an intriguing find, because only one other enzyme in plants is known to use nickel in its catalytic centre, namely urease. To investigate the role of these different isoforms in plants, fluorescent fusion proteins were engineered to study their subcellular localization. Analysis oftransfected cells by confocal microscopy revealed that these three fusion proteins are targeted to different subcellular compartments, two to the cytosol and one to the chloroplast. The three genes also responded differently to several treatments at the level of gene expression, suggesting they may have distinct functions in metabolism. One isoform in particular was dramatically upregulated by soluble sugars, consistent with a role in detoxifying by-products of glycolysis. The suggested interpretation of these results is that GLXI was subject to an ancient gene duplication event that gave rise to zinc- and nickel-dependent forms of the enzyme in an early organism, prior to divergence of the three domains of life. Most modem organisms have evidently retained only one form of this enzyme, whereas plants are highly unusual amongst eukaryotes in possessing both zinc- and nickel dependent forms of the enzyme.EThOS - Electronic Theses Online ServiceGBUnited Kingdo