*Article* **Characteristics of a Novel Manganese Superoxide Dismutase of a Hadal Sea Cucumber (***Paelopatides* **sp.) from the Mariana Trench**

**Yanan Li 1,2, Xue Kong 1,2 and Haibin Zhang 1,\***


Received: 17 December 2018; Accepted: 15 January 2019; Published: 1 February 2019

**Abstract:** A novel, cold-adapted, and acid-base stable manganese superoxide dismutase (Ps-Mn-SOD) was cloned from hadal sea cucumber *Paelopatides* sp. The dimeric recombinant enzyme exhibited approximately 60 kDa in molecular weight, expressed activity from 0 ◦C to 70 ◦C with an optimal temperature of 0 ◦C, and resisted wide pH values from 2.2–13.0 with optimal activity (> 70%) at pH 5.0–12.0. The *K*m and *V*max of Ps-Mn-SOD were 0.0329 ± 0.0040 mM and 9112 ± 248 U/mg, respectively. At tested conditions, Ps-Mn-SOD was relatively stable in divalent metal ion and other chemicals, such as β-mercaptoethanol, dithiothreitol, Tween 20, Triton X-100, and Chaps. Furthermore, the enzyme showed striking stability in 5 M urea or 4 M guanidine hydrochloride, resisted digestion by proteases, and tolerated a high hydrostatic pressure of 100 MPa. The resistance of Ps-Mn-SOD against low temperature, extreme acidity and alkalinity, chemicals, proteases, and high pressure make it a potential candidate in biopharmaceutical and nutraceutical fields.

**Keywords:** expression; purification; deep-sea enzyme; pCold vector

#### **1. Introduction**

Reactive oxygen species (ROS) are necessary for various physiological functions, such as signaling pathways and immune responses; the mass accumulation of ROS will damage bio-macromolecules, leading to cell death and various diseases [1,2]. Superoxide dismutases (SODs, EC 1.15.1.1) are one of the most important antioxidant enzymes that clear ROS by converting them into oxygen and hydrogen peroxide. According to the different metal cofactors, several types, such as Cu,Zn-SOD, Mn-SOD, Fe-SOD, cambialistic SOD (activated with either Fe or Mn), Ni-SOD, and Fe,Zn-SOD, have been reported in many species [3–7].

Studies have shown that SODs are related to immune reactions in invertebrates, as exemplified by bacterial and viral invasion [4,8], environmental pollution [9,10], and temperature stimulation [11]. Recently, Xie et al. indicates that antioxidant is related to the deep-sea environmental adaptability [12]. On the other hand, point mutations and activity loss of SODs lead to serious diseases and death in vertebrates. For example, the mice model of mitochondria SOD-deficiency is characterized by neurodegeneration, myocardial injury, and perinatal death [13,14]. A strong link is observed between Alzheimer's disease, tumor, amyotrophic lateral sclerosis, and SODs [15,16]. Hence, the physiological significance of SODs allows their application in the therapeutic and nutraceutical fields. To date, SODs have been reported to exhibit positive effects on inflammatory diseases, arthritis tumor, and promotion [17–19]. An orally effective form of SOD (glisodin) has been developed by Isocell Pharma, and it showed cosmetic and health benefits in human subjects [20,21]. Producing SOD using engineered

bacteria is one of the most promising methods to obtain high yield and inexpensive SODs for application. Therefore, the development of SODs with remarkable characteristics is particularly urgent.

Sea cucumbers are highly important commercial sea foods owing to their high nutritional value, and they are distributed from shallow water to the deep sea [22]. Although deep sea is an extremely low-temperature and high hydrostatic-pressured environment for most living organisms, holothurians dominate benthic megafaunal communities in hadal trenches and form "the kingdom of Holothuroidea" when food is abundant [23]. Extreme environments, such as the deep sea, are ideal for the development of new enzymes; numerous novel enzymes with unique activities, such as proteases and lipases, have been identified from the deep sea [24,25]. Considering the promising applications of SODs in therapeutic and nutraceutical fields, relationship with the adaptability of the deep-sea environment and limited studies in extreme organisms, especially in hadal sea cucumbers, we report a novel manganese superoxide dismutase from hadal sea cucumber *Paelopatides* sp. (Ps-Mn-SOD), which inhabits a depth of 6500 m in the Mariana Trench, analyzed its biochemical characteristics, and evaluated its stability for potential use in the food and preliminarily nutraceutical fields.

#### **2. Results**

#### *2.1. Sequence Characteristics*

The ORF of Ps-Mn-SOD is 768 bp long, encoding 255 amino acids. A signal peptide was detected at the N-terminal of deduced amino acid sequence. The N- and C-terminal domains spanned from Lys-34 to Ser-127 and Pro-137 to Leu-242, respectively. Four conserved amino acid residues, namely, His-63, His-119, Asp-209, His-213 are responsible for manganese coordination. A conserved residue of Tyr-35 is responsible for the second coordination sphere of the metal [26]. A highly conserved Mn-SOD signature sequence with the pattern D-x-[WF]-E-H-[STA]-[FY] existed in Ps-Mn-SOD (DVWEHAYY). The predicted secondary structure contained 13 α-helices and 4 β-strands. The deduced theoretical isoelectric point was 5.05, and the molecular weight was 29.29 kDa. The instability index of 36.97 classified the protein as stable. The 3D model of Ps-Mn-SOD was predicted using the x-ray template of *Bacillus subtilis,* which shared 45.27% sequence identity (PDB ID: 2RCV) [27]. This model shows that Ps-Mn-SOD is presented as a homodimer, and each subunit embraces one manganese ion. The global and per-residue model qualities were assessed using the QMEAN scoring function [28]. GMQE and QMEAN4 Z-scores reached 0.64 and −2.63, respectively, suggesting the accuracy of predicted 3D model of Ps-Mn-SOD. Figure 1 and Supplementary Figure S1 provide the related structural information of Ps-Mn-SOD.

**Figure 1.** Nucleotide and corresponding amino acid sequences of Ps-Mn-SOD. The signal peptide is drawn with a red line. The signature sequence DVWEHAYY is underlined with dotted line. N- and C-terminal domains are marked with purple and green shades, respectively. Four conserved amino acid residues for manganese coordination are boxed. Asterisk points to the highly conserved Tyr-35 residue. Cylinders and arrows represent helices and strands, respectively.

#### *2.2. Homology and Phylogenetic Analysis*

Multiple alignment and pairwise homology analysis between Ps-Mn-SOD and other invertebrates were performed, and the results are shown in Figure 2 and Supplementary Table S1. Multiple alignment of Ps-Mn-SOD with other invertebrates indicated that four amino acids were responsible for manganese binding, and the signature sequences are highly conserved in different Mn-SOD sources and were also identified in Ps-Mn-SOD (Figure 2). The highest similarity and identity were shared with *Apostichopus japonicus* (83.9% and 78.0%), followed by *Capitella teleta* (66.9% and 47.9%), *Exaiptasia pallida* (66.3% and 47.7%), *Strongylocentrotus purpuratus* (65.1% and 47.0%), *Mizuhopecten yessoensis* (64.4% and 46.7%), and *Stylophora pistillata* (63.1% and 45.8%). To determine the type of SOD present, we performed phylogenetic analysis based on the amino acid sequences of the determined SOD types in Genebank (Figure 3). The results showed that the present SOD clustered with *A. japonicus* and evidently a Mn-SOD type with high bootstrap values.

**Figure 2.** Multiple alignment of Ps-Mn-SOD with other invertebrates. Mn-SOD signature sequence is boxed. Triangles point to the active sites for manganese coordination. Asterisk points to the highly conserved Tyr-35 residue.

**Figure 3.** Neighbor-joining phylogenetic tree of SODs based on amino acid sequence homology. Bootstrap values below 50 are cut off. Ps-Mn-SOD is displayed in bold.
