Elsevier

Biochimie

Volume 97, February 2014, Pages 181-193
Biochimie

Research paper
Mechanistic study of CuZn-SOD from Ipomoea carnea mutated at dimer interface: Enhancement of peroxidase activity upon monomerization

https://doi.org/10.1016/j.biochi.2013.10.014Get rights and content

Highlights

  • Monomeric Ipomoea carnea CuZn-SOD was made by a single amino acid substitution.

  • The absorption spectrum of IcSOD shows a novel absorbance in 370–400 nm region.

  • Monomerization causes enhancement of peroxidase with lowering of SOD activity.

  • Higher degree of temperature susceptibility was observed in the monomeric CuZn-SOD.

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.

Introduction

Superoxide dismutases (EC 1.15.1.1), belong to a family of metallo-enzymes, which catalyze the disproportionation of superoxide anion radical (O2radical dot) in a two-step reaction kinetics resulting in formation of molecular oxygen (O2) and H2O2 [1]. Therefore, SODs play an important protective role in cells by preventing the reactive oxygen species (ROS) mediated oxidation of cellular components. Based on the presence of metal ion cofactor(s) in their active site, SODs are classified as CuZn, Mn, Fe, Ni, cambialistic (Fe/Mn or Fe/Zn) SODs, and heme co-ordinated SOD [2]. The latter three forms are, however, reported in prokaryotes only [3]. Iso-forms of CuZn-SOD, the most abundant SOD in higher plant cells are found in chloroplast, cytosol, peroxisome, and in extra cellular space [3], [4].

Eukaryotic CuZn-SODs are in general homodimer and non-covalently bonded by identical sub-units containing a copper (Cu2+) and a zinc (Zn2+) ion [5]. The oxidized copper ion (Cu2+) serves as the redox partner of O2radical dot, whereas Zn2+ appears to stabilize the dimer assembly and plays a role in electrostatic stabilization of the enzyme [6]. Based on the observation that the native sub-units exhibit identical activity, it has been suggested that the active sites in each of the sub-units can function independently [7]. In addition to the presence of dimeric forms, natural occurrence of monomeric form of CuZn-SOD, like those found in bacterial system is also reported in plant species [8], [9], suggesting physiological relevance of the monomeric structure of the enzyme. Prokaryotic CuZn-SOD shows larger structural variability than eukaryotic enzymes in terms of their active site channel organization and arrangement of amino acid residues at dimer interface [10].

In humans, mutations in the dimeric interface of CuZn-SOD (SOD 1) that cause destabilization of the quaternary structure of dimeric CuZn-SOD and eventually lead to misfolding or formation of aggregates that may lead to fALS disease [11]. These mutant SODs generally retain their full activity; however there is a toxic gain of oxidative function. Another interesting feature that has attracted some of these aberrant CuZn-SODs for investigation is the stimulation of disproportionation of O2radical dot anion along with additional peroxidative activity [12], [13]. Hence, these results provide rationale to study of peroxidase action of the CuZn-SODs and their mutant form, both under in vitro and in vivo conditions.

Ipomoea carnea (I. carnea) subsp. fistulosa of the Convolvulaceae family (morning glory) is a toxic plant. The toxic principles of the plant have been identified as two nortropane alkaloids, calystegines B2 and C1 and an indolizidine alkaloid swainsonine [14]. The plant serves as one of the most suitable species for phyto-extraction of cadmium from soil [15]. However, the plant has not been thoroughly examined to understand its antioxidative potential. Although, different species of Ipomoea i.e., Ipomoea batata shows few interesting facts about the structure and functional aspects of the enzyme [16], there is dearth of information on antioxidative enzymes of I. carnea, that grows abundantly in all kinds of agronomic conditions.

In the present study, the structure–function relationship of dimeric recombinant I. carnea CuZn-SOD and its monomeric variant have been explored. The monomeric form of the protein was obtained by site directed mutagenesis of one leucine (Leu) with a charged lysine (Lys) residue. The resulting dimer showed low stability with decrease in superoxide scavenging activity. However, there was an increase in peroxidase activity in mutated form as compared to wild type. These observations led us to perform detailed molecular modeling studies that connect different experimental observations of the mutant protein and revealed the characteristic structural changes of the protein, leading to disruption of residue–residue contacts and monomerization of the native protein.

To the best of our knowledge this is the first report, where peroxidase activity of a plant CuZn-SOD has been studied in vitro and further extended to its monomeric variant. The present study has re-examined the role of subunit–subunit interactions to decipher the stability and catalytic properties of the enzymes. In this report, the wild type I. carnea CuZn-SOD (IcSOD) has been described as Wild Type or WT, while the mutant one as mutant or MT or variant.

Section snippets

Plant material and chemicals

The leaves of I. carnea that grows widely in the Institute of Life Sciences campus, Bhubaneswar, India were collected, washed thoroughly under tap water followed by distilled water before use in the experiments.

Unless otherwise indicated, all analytical grade reagents were procured from Sigma. The expression vector pQE30-UA, Escherichia coli M15 cells, Ni-NTA columns and matrix were procured from Quiagen. DNA modifying enzymes, Taq DNA polymerase, AMV reverse transcriptase, pGEM T-Easy vector

Overexpression, purification and molecular weight determination

The IcSOD was recombinantly expressed in E. coli and purified to its homogeneity. The yield of the purified protein varied between 4 and 5 mg L−1 of culture volume (bacterial wet biomass was about 4.0–4.5 g L−1). Inclusion of Cu (copper sulfate) and Zn (zinc sulfate) in the growth media and/or reconstitution of the purified proteins with these metals did not cause any alteration in protein yield, electronic transition spectral characteristics and specific activity of the protein. Gel filtration

Discussion

The enzymatic function of a protein is a co-ordinated event of its physico-chemical properties. The different states of oligomerization govern its stability, while the chemistry of its substrate affinity determines the true catalytic activity. Though, CuZn-SOD has been thoroughly characterized in terms of their structure–function relationship, occurrence of both monomeric and dimeric form of the enzymes in both prokaryotes and eukaryotes raise fundamental question about the role of quaternary

Conclusion

In essence, the dimeric organization of eukaryotic CuZn-SODs is prime requirement for its stability and conservation of the redox center and any disarrangement in its oligomeric organization will lead to alteration in stability and the catalytic efficiency of the enzyme. These forms although have no significant differences in metal content and electronic transition properties, they undergo significant alteration in the specific activity related to scavenging of O2radical dot and H2O2. We believe that the

Acknowledgments

SCS would like to dedicate this work to his Graduate Teacher, Late Professor Shaymapada Saha, Kalyani University, West Bengal, India. The authors are thankful to The Director, Institute of Life Sciences for providing chemical and consumables support. We are grateful to Prof. Raghavan Varadarajan and Dhabaleswar Patra, Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India for providing the major instrumental facility like CD, DSC and for MASS analysis, reported in this work.

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