Comprehensive characterization and molecular insights into the salt tolerance of a Cu, Zn-superoxide dismutase from an Indian Mangrove, Avicennia marina

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.

Figure 1

A. marina displayed highest SOD activity among 3 species. (a) Photograph of twigs with inflorescence of 3 species of Avicennia collected from Sundarban mangrove. Horizontal yellow bar represents 10 cm. (b) SOD activity assay from leaf extract of these 3 species. SOD activity is expressed as unit of enzyme present in each gram of leaf tissue converting the substrate into product in a minute plotted in y-axis. Each bar graph represents the mean of 6 biological replicates collected from 6 different locations (n = 6) and error bar as SD.

Figure 2

Purification of Am_SOD from A. marina leaf. Supernatant fraction after 60% ammonium sulfate cut of leaf extract was fractionated in anion exchange column followed by gel filtration. Chromatograms are shown in (a) and (d). The A₂₈₀ of eluted fractions are in y-axis versus elution volume in x-axis. Void volume (9.2 ml) of the gel filtration column is demarcated by dashed line in (d). SOD activity assay (in y-axis) was done to screen for the presence of Am_SOD in each column eluted fraction (in x-axis) as shown in (b). Fraction number 5 (Fr-5) of anion exchange chromatography showed highest SOD activity and partially purified Am_SOD protein in SDS-PAGE (c). Fr-5 was re-fractionated in gel filtration column and Fr-5B displayed highest SOD activity (e) with purified Am_SOD in > 85% homogeneity in SDS-PAGE (f).

Figure 3

Recombinant Am_SOD is a functionally active superoxide dismutase. (a) 6xHis-tagged Am_SOD was purified by Ni–NTA chromatography in soluble form. Lane U; uninduced control, lane I; supernatant fraction after sonication of IPTG-induced E. coli cells harboring Am_SOD-pET22b+ construct. Flow-through (lane FT) from supernatant fraction after binding with Ni–NTA. Beads were washed twice (lanes W1 and W2) with 40 mM imidazole. Column-bound Am_SOD was eluted (lane E) with 250 mM imidazole. Left margin (M) is MW marker. (b) 12% SDS-PAGE showing Am_SOD dimer (~ 32 kDa) under non-reducing condition without β-ME (lane NR) and monomer (~ 16 kDa) under reducing condition with β-ME (lane R). Right margin (M) is MW marker. (c) Absorption spectra of 0.8 mg/ml of Am_SOD at 300–800 nm (in x-axis) wavelengths showing an electronic transition at 380–400 nm region which is magnified and shown in inset. Electronic transition suggests an interaction of Cu²⁺ with imidazole ring of His-62. (d) Michaelis–Menten kinetics curve showing specific activity of Am_SOD (0.005 mg/ml) expressed as unit of enzyme per milligram of protein (in y-axis) as a function of riboflavin concentrations (µM, in x-axis). (e) Lineweaver–Burk plot showing linear regression of Am_SOD kinetics. [V] and [S] are reaction velocity (y-axis) and substrate concentrations (x-axis) respectively. Linear relationship (R²) and straight-line equation of the plot are shown. V_max and K_m were determined as inverse of y- and x-intercepts respectively. (f) Zymography in native PAGE showing SOD activity where 1 µg Am_SOD formed a hyaline zone and rest of the gel turned blue due to NBT oxidation by superoxide radicals generated from riboflavin. Dose- dependent inhibition of specific activity of 0.005 mg/ml of Am_SOD by increasing concentrations (in x-axes) of Sodium diethyldithiocarbamate trihydrate/DDC (g) and hydrogen peroxide/H₂O₂ (h). The SOD activity is presented here as a percentage of activity (in y-axis) at a certain inhibitor concentration after a 0.5 h of incubation with respect to the maximum activity at 0 h at that particular inhibitor concentration. (i) and (j) showing zymography of dose-dependent inhibition of 1 µg of Am_SOD activity incubated with increasing concentrations of DDC and H₂O₂ respectively.

Figure 4

Studying salt tolerance of Am_SOD by fluorescence spectrometry. (a) Fluorescence spectra showing tyrosine autofluorescence intensity (in y-axis) of 0.05 mg/ml of Am_SOD incubated with various NaCl concentrations and scanned at emission wavelength from 290 to 400 nm (in x-axis). A plot in the inset showing no noticeable change in normalized fluorescence intensity of Am_SOD (ratio between intensities at 305 to 315; in y-axis) at increasing NaCl concentration (in x-axis). (b) and (c) showing modified Stern–Volmer plots of tyrosine fluorescence quenching of Am_SOD by increasing concentrations (in x-axis) of acryalmide and iodide respectively. In each quenching experiment, Am_SOD was incubated with 500 mM NaCl or without salt treatment (control). Fluorescence quenching data is presented here on y-axis as a ratio between fluorescence intensity without quencher (F0) and difference in fluorescence intensity after adding quencher (ΔF). The calculated values of quenching parameters are displayed in the tables adjacent to each Stern–Volmer plot. (d) Plot showing surface hydrophobicity in terms of fluorescence emission spectra at 490 nm (in y-axis) of c treated with or without NaCl and then titrated with increasing concentrations (in x-axis) of Bis-ANS.

Figure 5

Impact of salt and pH on Am_SOD activity. (a) Plot of single light scattering experiment showing resistance of Am_SOD (i) to NaCl-induced aggregation. No significant increase in absorbance of salt-treated versus untreated Am_SOD at 360 nm (in y-axis) was observed over time (in x-axis). A salt-sensitive profilin protein, Sola m 1 from eggplant (ii) was used as control. (b) and (c) Plots showing specific activity (in y-axis) of 0.005 mg/ml of Am_SOD in presence of increasing concentrations of NaCl and 4 different pH values (in x-axes) respectively. 1.17 µM riboflavin was used as substrate in all reactions. Each data point is a mean of triplicate measure and SD as error bars. (d) Functional complementation of Am_SOD in E. coli on LB-agar plates with 500 mM NaCl (salt treated) and with 0 mM salt (untreated control). Both the plates were supplemented with methyl viologen to induce oxidative stress, IPTG for protein induction, and ampicilin for selection. Appearance of growth was observed for sod double mutant strain QC774 transformed with Am_SOD construct (area 1) on both the plates suggesting the ability of Am_SOD to remain functionally active for combating oxidative stress under high saline condition. K12 strain with functional native sod genes harboring pET22b+ vector (area 2) grew only in zero salt plate (under oxidative stress only). QC774 strain harboring pET22b+ vector (area 3) failed to grow under oxidative as well as salinity stress.

Figure 6

Am_SOD displayed certain degree of heat tolerance. (a) CD spectra showing molar ellipticity (in x-axis) of 5 µM of Am_SOD at wavelengths 200–260 nm (in x-axis) and at 25 °C. (b) Step-scan showing raw CD millidegrees (in y-axis) of 5 µM of Am_SOD at wavelengths 200–260 nm (in x-axis) within a temperature range from 25 to 90 °C with a 5 °C increment. After 90 °C, CD spectra were recorded once again at 25 °C. (c) Melting curve of Am_SOD showing no noticeable change in the fractions of α-helices and β-sheets present in the protein (ratio of CD millidegree at 222 and 216 nm, in y-axis) in an ascending scan or AS (step-wise from 25 to 90 °C) as well as in a descending scan or DS (direct from 90 to 25 °C). Melting curve of sunflower pectate lyase Hel a 6 (Ha_PL), a heat-sensitive control protein is also plotted for comparison. Ha_PL showed reversible thermal denaturation in which the protein was fully unfolded at 90 °C but partially refolded upon cooling. (d) Effect of temperature (in x-axis) on specific activity of 0.005 mg/ml of Am_SOD. The SOD activity is presented here as a percentage of activity (in y-axis) at a certain temperature with respect to the SOD activity at 37 °C (considered as optimum activity). 1.17 µM riboflavin was used as substrate in each reaction. Each data point is a mean of triplicate measure and SD as error bars.

Figure 7

Mapping of critical residues conferring salt tolerance to Am_SOD by in silico studies. (a) Multiple sequence alignment of amino acid sequences of Am_SOD with 3 non-halophilic Cu, Zn-SOD proteins from Potentilla (Pa_SOD), tomato (Sl_SOD), and tobacco (Nt_SOD) using Clastal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/). Identical residues on aligned sequences are shown in asterisks. 5 N-terminal residues in Am_SOD with decreased hydrophobicity as compared to the non-halophilic counterparts are highlighted in green. The highly conserved catalytic domain is underlined. The cysteine residue responsible for dimer formation is shown in red and the histidine residues responsible for metal ligand (Cu and Zn) binding are shown in yellow. (b) The homology model of Am_SOD dimer built in SWISS-MODEL server (https://swissmodel.expasy.org/) shown as cartoon and surface representation for chain-A and chain-B respectively using PyMol v2.5 (https://pymol.org/2/). Atomic structure of those 5 critical residues is labeled on chain-A. (c) Plot showing changes in hydropathy index values (in y-axis) in these 5 critical residues of Am_SOD as compared to corresponding residues in the same position (in x-axis) on 3 non-halophilic Cu, Zn-SOD proteins.

Figure 8

Mutation in 5 N-terminal critical residues resulted in decrease in salt tolerance of Am_SOD. (a) Plot showing specific activity (in y-axis) of wild type/WT Am_SOD and 6 mutant versions under various NaCl concentrations (in x-axis). Significant reduction (p < 0.05 as asterisk) in SOD activity was observed in all the 6 mutants at 500 mM NaCl where the maximum reduction (~ 54%) was observed for the multiple-point mutant harboring all the 5 point mutations. (b) and (c) Plots of single light scattering experiment showing NaCl-induced aggregation of the multiple-point mutant versus resistance to NaCl-induced aggregation of Am_SOD respectively. Here, 3 different NaCl concentrations were used to treat the proteins and absorbance at 360 nm (in y-axis) was scanned at different time points (in x-axis).