Characterization of recombinant alternative Oxidase 1A from arabidopsis thaliana and interaction with inhibitors TCA cycle and redox metabolites

Abstract

Abstract of Thesis The plant mitochondrial electron transport chain possesses two terminal oxidases, cytochrome oxidase (COX) and alternative oxidase (AOX) pathways for the reduction of molecular oxygen into water. The COX and AOX pathways are known to bifurcate at ubiquinone. The AOX pathway is identified in all plants, fungi, some protists, and a few animal and bacterial species but is absent in mammals, and this pathway is not linked to ATP generation. Though the AOX pathway was initially thought to be an energy-wasteful process, several studies from the past three decades revealed its importance in regulating cellular redox, ROS and metabolic homeostasis under various biotic and abiotic stresses. The alternative oxidase1A (AOX1A) from Arabidopsis thaliana is the most important among hundreds of known stress-responsive genes of plant mitochondria. Arabidopsis thaliana AOX1A is present in the mitochondrial inner membrane towards the matrix side. Despite its important role in cellular redox and metabolic homeostasis, the mechanism of its interaction with different metabolites is poorly understood due to the difficulty in acquiring it in a pure and highly active form of protein from plant sources. Therefore, in the present study, the AtAOX1A from Arabidopsis thaliana is cloned into a pET28a vector and induced its expression in E. coli using IPTG. The expressed protein is functionally active in E. coli, which is evident by KCN (inhibitor of COX pathway) resistance and salicylhydroxamic acid (SHAM) and n-propyl gallate (n-PG) [inhibitors of AOX pathway] sensitive respiration and growth. Later, the rAtAOX1A is purified in its active form from E. coli membranes by solubilizing in DDM and passing through a cobalt affinity column. The purified rAtAOX1A has shown stability in its structure to a wide range of temperature and pH conditions. newlineIn the literature, it has been reported that AOX isoforms are posttranslationally activated by different TCA cycle metabolites in an isoform-specific manner. Therefore, we made an atte