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Mar Drugs
2018 May 18;165:. doi: 10.3390/md16050169.
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Characteristics of the Copper,Zinc Superoxide Dismutase of a Hadal Sea Cucumber (Paelopatides sp.) from the Mariana Trench.
Li Y
,
Kong X
,
Chen J
,
Liu H
,
Zhang H
.
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Superoxide dismutases (SODs) are among the most important antioxidant enzymes and show great potential in preventing adverse effects during therapeutic trials. In the present study, cloning, expression, and characterization of a novel Cu,Zn superoxide dismutase (Ps-Cu,Zn-SOD) from a hadal sea cucumber (Paelopatides sp.) were reported. Phylogenetic analysis showed that Ps-Cu,Zn-SOD belonged to a class of intracellular SOD. Its Km and Vmax were 0.0258 ± 0.0048 mM and 925.1816 ± 28.0430 units/mg, respectively. The low Km value of this enzyme represents a high substrate affinity and can adapt to the low metabolic rate of deep sea organisms. The enzyme functioned from 0 °C to 80 °C with an optimal temperature of 40 °C. Moreover, the enzyme activity was maintained up to 87.12% at 5 °C. The enzyme was active at pH 4 to 12 with an optimal pH of 8.5. Furthermore, Ps-Cu,Zn-SOD tolerated high concentration of urea and GuHCl, resisted hydrolysis by proteases, and maintained stability at high pressure. All these features demonstrated that the deep sea Ps-Cu,Zn-SOD is a potential candidate for application to the biopharmaceutical field.
Figure 1. Nucleotide and deduced amino acid sequences of Paelopatides sp. copper, zinc superoxide dismutase (Ps-Cu,Zn-SOD). Conserved amino acid residues for Cu-binding are underlined, whereas residues for Zn-binding are shaded in green. Two cysteines predicted to form a disulfide frame are boxed. Cylinders and arrows represent helices and strands, respectively. The shaded part represents the predicted domain area.
Figure 2. Multiple alignment of Ps-Cu,Zn-SOD with other Echinodermata and invertebrates. Identities and similarities are shaded in black and gray, respectively. The predicted eight β-strands, two α-helices, and two sequences of Cu,Zn-SOD family (yellow box, ps1 and ps2) are shown above the alignment. Amino acids for Cu- and Zn-binding are indicated with triangles and asterisks. The two cysteines forming a disulfide bond are indicated with plus signs. Information on the species used for the alignment is given in Table 1.
Figure 3. (A) Comparison of predicted structure with non-redundant set of PDB (Protein Data Bank) structures. The large black dots represent QMEAN Z-score between −1 and 1. The red star represents the model. (B) The four individual terms of the global QMEAN quality scores. (C) Predicted 3D structure of Ps-Cu,Zn-SOD using the Swiss model. Pink and purple spheres represent zinc and copper ions, respectively. The protein is a homodimer. N and C represent the N- and C-terminals of the polypeptide chain. (D) Close-up of the active sites.
Figure 4. Neighbor-joining phylogenetic tree of Ps-Cu,Zn-SOD and Cu,Zn-SOD amino acid sequences from different species. The tree was built using MEGA7 and bootstrap was set as 1000. The star sign represents the position of Ps-Cu,Zn-SOD in the phylogenetic tree.
Figure 5. Analysis of the expressed protein via SDS-PAGE. M: protein marker, Lane 1: total proteins from E. coli pG-KJE8/BL21 containing recombinant plasmid before IPTG induction, Lane 2: total proteins from E. coli pG-KJE8/BL21 containing recombinant plasmid after IPTG induction, Lane 3: inclusion body after ultrasonication, Lane 4: supernatant after ultrasonication, Lane 5: recombinant protein purified using Ni2+-NTA column, Lane 6: Western blot of recombinant protein.
Figure 6. (A). Effect of temperature ranging from 0–80 °C on the enzyme activity. Each square in the graph represents the mean ± SD (n = 3). (B) Effect of pH on the enzyme activity. Tests were performed using different pH buffers ranging from 3.0–12.0 °C at 25 °C for 1 h. Each square in the graph represents the mean ± SD (n = 3). (C) Effects of denaturants on SOD activity. Values are mean ± SD of three separate replicates. The residual enzyme activity of the sample without denaturant was defined as 100%. (D) Effects of high hydrostatic pressure on SOD activity. Enzyme activity at 0.1 MPa at 5 °C was considered as 100%. Data are shown as mean values (n = 3) ± SD. (E) Michaelis-Menten equation of recombinant Ps-Cu,Zn-SOD. * p < 0.05; ** p < 0.01.
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