Elucidating Mechanisms for S-Nitrosoglutathione Reductase Activity and Control in Plants Using O-Aminobenzoyl-S-Nitrosoglutathione and a Novel Photo-Sensitive Probe
Date of Award
Chemistry and Biochemistry
Photo-sensitive probe, S-nitrosoglutathione reductase, Solanum lycopersicum, Ammonium, Allosteric site
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S-nitrosoglutathione reductase, (GSNOR) is widely accepted as the master regulator of stress through NO signaling and protein S-nitrosylation. GSNOR mediates stress response through the catalysis of its principal substrate S-nitrosoglutathione (GSNO). The instigation of various stressors in plants cause observable changes in plant phenotype, which are associated with changes in GSNOR activity. There are no current methods for measuring GSNOR activity directly in living plants. In this paper, a previously developed fluorogenic pseudo-substrate for human GSNOR, OAbz-GSNO, was applied to the Solanum lycopersicum plant model. OAbz-GSNO was identified as a promising novel pseudo- substrate to study changes in GSNOR activity in the presence of stressors, using fluorescence microscopy and living plant tissue. Ammonium is one of the most prevalent nitrogen sources available, however, ammonium becomes toxic to plants at over 0.5mM. GSNOR activity was assessed in roots grown in high ammonium stress conditions where activity was shown to decrease despite an up-regulation in GSNOR synthesis. These results suggest that GSNOR can also be controlled through post-translational modifications.
Previous studies have identified a possible modification at an allosteric site at residues Lys323, Gly321, Asn185, and Lys188, which may cause an increase in activity at high GSNO concentration. A photo-liable affinity probe, p-azidophenacyl glutathione (papaGS) was designed to interact with GSNO binding sites. In dark conditions, enzyme kinetic experimentation identified a reduction in cooperativity binding when HmGSNOR is exposed to papaGS, indicating specificity to the allosteric site. When irradiated the HmGSNOR function was also inhibited to 50% activity by azide non-specific crosslinking at the allosteric site causing a conformational change, indicating the importance of the site in proper enzyme function and the usefulness of papaGS as an allosteric site probe for GSNOR.
Ventimiglia, Leslie, "Elucidating Mechanisms for S-Nitrosoglutathione Reductase Activity and Control in Plants Using O-Aminobenzoyl-S-Nitrosoglutathione and a Novel Photo-Sensitive Probe" (2022). Electronic Theses and Dissertations. 8976.