Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8305875 | Biochimie | 2014 | 13 Pages |
Abstract
The enzymatically active monomeric form of CuZn-superoxide dismutase has always been of interest to decipher the structure-function relationship in this class of enzymes. In the present study, spectroscopic and enzymatic characteristics of the dimeric and monomeric forms of recombinant Ipomoea carnea CuZn-superoxide dismutase were made to decipher their stability and altered catalytic properties. The monomeric form of protein was produced through site directed mutagenesis by replacing a conserved hydrophobic leucine with a polar lysine residue at the dimer-interface. Spectral characteristics of both the forms (monomer and dimer) showed the presence of novel electronic transitions. Superoxide scavenging activity of the mutated form was reduced to nearly half of the activity found in the native enzyme. Concomitantly, compared to native form the mutated enzyme showed an increase in peroxidase activity. High temperature dependent circular dichroism spectral analysis, differential scanning calorimetric profile, and the measurement of temperature dependent superoxide scavenging activity indicated an increased susceptibility of the mutated form to higher temperature as compared to the native form. The inhibitor studies like hydrogen peroxide, diethyldithiocarbamate and phenylglyoxal also indicate higher susceptibility, which might be due to, altered arrangement of active site residues as a consequence of the mutation. Molecular modeling and MD simulation studies further indicated that this specific mutation induces loss of hydrophobic interaction at dimer interface, resulting in the observed instability of the dimeric form. Increased peroxidative activity of the enzyme, upon monomerization may have physiological implication essentially in presence of high concentration of H2O2, as in case of plant cells specifically under stress conditions.
Keywords
DSC2′,7′-dichlorfluorescein-diacetateNaN3PGXDCFH-DATFARMSDIPTGDTPABSAddCKCNHydrogen peroxidebovine serum albuminTrifluoroacetic acidisopropyl β-D-1-thiogalactopyranosidefALSMelting TemperatureDiethylenetriamine pentaacetic aciddiethyldithiocarbamateSODsodium azideSuperoxide dismutaseMD simulationinductively coupled plasma mass spectrometryICP-MSroot mean square distanceSuperoxide dismutase activityperoxidase activityPhenylglyoxalMALDI-TOF-MSH2O2potassium cyanideDifferential scanning calorimetry
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Authors
Panchanand Mishra, Anshuman Dixit, Mamata Ray, Surendra Chandra Sabat,