Abstract
Superoxide dismutases are important group of antioxidant metallozyme and play important role in ROS homeostasis in salinity stress. The present study reports the biochemical properties of a salt-tolerant Cu, Zn-superoxide from Avicennia marina (Am_SOD). Am_SOD was purified from the leaf and identified by mass-spectrometry. Recombinant Am_SOD cDNA was bacterially expressed as a homodimeric protein. Enzyme kinetics revealed a high substrate affinity and specific activity of Am_SOD as compared to many earlier reported SODs. An electronic transition in 360–400 nm spectra of Am_SOD is indicative of Cu2+-binding. Am_SOD activity was potentially inhibited by diethyldithiocarbamate and H2O2, a characteristic of Cu, Zn-SOD. Am_SOD exhibited conformational and functional stability at high NaCl concentration as well in alkaline pH. Introgression of Am_SOD in E. coli conferred tolerance to oxidative stress under highly saline condition. Am_SOD was moderately thermostable and retained functional activity at ~ 60 °C. In-silico analyses revealed 5 solvent-accessible N-terminal residues of Am_SOD that were less hydrophobic than those at similar positions of non-halophilic SODs. Substituting these 5 residues with non-halophilic counterparts resulted in > 50% reduction in salt-tolerance of Am_SOD. This indicates a cumulative role of these residues in maintaining low surface hydrophobicity of Am_SOD and consequently high salt tolerance. The molecular information on antioxidant activity and salt-tolerance of Am_SOD may have potential application in biotechnology research. To our knowledge, this is the first report on salt-tolerant SOD from mangrove.
Highlights
Oxidative stress in aerobic organisms refers to the production of oxygen byproducts and is triggered by various environmental factors
The present study presents a comprehensive characterization of a novel Superoxide dismutase (SOD) enzyme isolated from a mangrove species of Indian origin using biochemical and biophysical methods
The kinetic data of Am_SOD represents a high substrate affinity and strong superoxide dismutation activity as compared to many previously reported Cu, Zn-SODs of eukaryotic origin. Such a robust activity of Am_SOD is thought to be the key for homeostasis of the exceptionally high level of reactive oxygen species (ROS) resulting from salinity stress and thereby protecting the cellular components from oxidative damage
Summary
Oxidative stress in aerobic organisms refers to the production of oxygen byproducts and is triggered by various environmental factors. The most immediate response to these environmental stresses is the enhanced level of free radicals that include singlet oxygen (1O2), hydrogen peroxide ( H2O2), superoxide radical ( O2−·), hydroxyl radical (OH·), hydroperoxyl/perhydroxyl radical (HO2·), alkoxy radical (RO·), peroxy radical (ROO·) and excited carbonyl (RO*). These reactive oxygen species (ROS) can lead to potential damage at cellular as well as genetic level leading to detrimental effects such as cell death and DNA mutation[1]. A detailed understanding of the molecular basis of the high antioxidant activity of this enzyme in presence of high salt concentration is not yet available In this communication, we report a full-length Cu, Zn-SOD enzyme (Am_SOD) isolated from the Avicennia marina of Indian Sundarban. We present here a deeper insight into salt-tolerant features of this enzyme at residue level through a rational mutagenesis approach
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