Homoglutathione Synthetase And The Plant Thiol-Redox Proteome

ABSTRACT OF THE DISSERTATION Homoglutathione Synthetase and the Plant Thiol-Redox Proteome by Ashley Galant Doctor of Philosophy in Biology and Biomedical Science: Plant Biology) Washington University in St. Louis, 2011 Professor Joseph M. Jez, Chairperson In the plant cell, redox regulation and redox responsiveness are governed by a series of mechanisms that hinge upon the use of small molecule redox-couples and reversible, thioredoxin- or glutaredoxin-mediated protein disulfides. This work examines first the structural basis for synthesis of these small molecules and second how plants are able to adapt and respond to changes in environmental redox state. Among the major redox-couples, glutathione: GSH) is maintained at the highest cellular concentrations, and is furthermore employed in a protective capacity as an anti-xenobiotic and anti-oxidation protein thiol-modification. Almost all eukaryotes utilize GSH, but some legumes additionally synthesize homoglutathione: hGSH), which is a GSH analog that contains a terminal beta-alanine residue instead of a terminal glycine. This alternate reaction is catalyzed by hGSH synthetase, which is related to GSH synthetase; however, the specific features that alter substrate specificity are unknown. To understand the molecular basis for the synthesis of the legume-specific molecule, the three-dimensional structure of hGSH synthetase from Glycine max: soybean) was solved by x-ray crystallography in three forms - apoenzyme, bound to y-glutamylcysteine, and with hGSH, ADP, and a sulfate ion bound in the active site. Comparison of these structures with those of GSH synthetase suggest that two residues - a leucine and a proline in the Ala-rich loop region of the enzymes - dictate the use of beta-alanine instead of glycine in hGSH synthetase. Site-directed mutagenesis studies and kinetic analysis further support this conclusion. As a means of regulating activity, many plant proteins limit access to their active sites and control the aggregation of catalytic oligomeric complexes through the formation of redox-reversible disulfide bonds. In order to identify plant proteins and pathways that utilize such bonds and/or thiol modifications to modulate oxidation state, an N-ethylmaleimide- and 5-iodoacetamidofluorescein-based dual-labeling strategy was employed in conjunction with 2D-gel electrophoresis and LC-MS/MS. Initial experiments with root protein extracts from B. juncea identified several new proteins that were differentially expressed and/or oxidized in response to exposure to the glutamate-cysteine ligase inhibitor buthionine sulfoxide or hydrogen peroxide. A clear lack of overlap between the proteins altered by each condition was also noted. To assess oxidative changes to the plant thiol-redox proteome under agriculturally relevant conditions, soybean plants were field grown under ambient and elevated tropospheric ozone concentrations. Investigation into changes in protein expression and oxidation state again yielded numerous novel protein responses. Intriguingly, many of the largest changes were observed in pathways involved in core carbon metabolism, a sharp contrast to the changes in redox-centric pathways seen following acute ozone exposure. This observation, in conjunction with a comparison of protein responses across several different ozone concentrations, led to the conclusion that ozone exposure is governed by a threshold effect: a concentration at which the plants transition from an active redox response toward maintenance of core processes and metabolism