Appl Biochem Biotechnol DOI 10.1007/s12010-014-0922-2
Comparative Analysis and Modeling of Superoxide Dismutases (SODs) in Brachypodium distachyon L. Ertugrul Filiz & Ibrahim Koc & Ibrahim Ilker Ozyigit
Received: 21 January 2014 / Accepted: 14 April 2014 # Springer Science+Business Media New York 2014
Abstract Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of superoxide radical to hydrogen peroxide and dioxygen. To date, four types of SODs — Cu/ZnSOD, MnSOD, FeSOD, and NiSOD — have been identified. In this study, SOD proteins of Brachypodium distachyon (L.) Beauv. were screened by utilization of bioinformatics approaches. According to our results, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found to be in basic and acidic character, respectively. Domain analyzes of SOD proteins revealed that iron/manganese SOD and copper/zinc SOD were within studied SOD proteins. Based on the seconder structure analyzes, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found as having similar sheets, turns and coils. Although helical structures were noticed in the types of Mn/ FeSODs, no the type of Cu/ZnSODs were identified having helical structures. The predicted binding sites of Fe/MnSODs and Cu/ZnSODs were confirmed for having His-His-Asp-His and His-His-His-Asp-Ser residues with different positions, respectively. The 3D structure analyzes of SODs revealed that some structural divergences were observed in patterns of SODs domains. Based on phylogenetic analysis, Mn/FeSODs were found to have similarities whereas Cu/ZnSODs were clustered independently in phylogenetic tree. Keywords Superoxide dismutase . Antioxidant proteins . Brachypodium distachyon . 3D modeling . In silico analysis
E. Filiz (*) Department of Crop and Animal Production, Cilimli Vocational School, Duzce University, 81750 Cilimli, Duzce, Turkey e-mail: [email protected] I. Koc Faculty of Science, Department of Molecular Biology and Genetics, Gebze Institute of Technology, Gebze, Kocaeli 41400, Turkey I. I. Ozyigit Faculty of Science and Arts, Department of Biology, Marmara University, 34722 Goztepe, Istanbul, Turkey
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Abbreviations SOD Superoxide dismutase FeSOD Iron superoxide dismutase MnSOD Manganese superoxide dismutase Cu/ZnSOD Copper/zinc superoxide dismutase ROS Reactive oxygen species Introduction Reactive oxygen species (ROS) are free radicals derived from O2 and are stimulated in various types of biotic and abiotic stresses. They can pose damages to biomolecules such as membrane and proteins at high concentrations whereas they can show functions as second messengers at low/moderate concentrations [1]. The most common types of ROS are hydrogen peroxide (H2O2), hypochlorite (OCl−), peroxynitrate (ONO2−), and hydroxyl radical (HO.), which can be removed by antioxidants and antioxidative enzymes. Major ROS-scavenging enzymes in plants are superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PrxR). SODs (EC 1.15.1.1) and CAT (EC 1.11.1.6) as ROS-scavenging systems save plant cells by decreasing the toxic forms of oxygen [2–4]. SODs (EC 1.15.1.1) are members of metalloenzymes catalyzing the dismutation of superoxide to oxygen and hydrogen peroxide [5]. Based on prosthetic metals, SODs can be categorized in four (canonical) groups: iron SOD (FeSOD), manganese SOD (MnSOD), copper/zinc SOD (Cu/ZnSOD), and nickel SOD (NiSOD). Although all types of SODs are found in prokaryotic organisms, eukaryotes only have three types: FeSODs (in chloroplasts), MnSODs (in mitochondria and peroxisomes), and Cu/ZnSODs (in chloroplast, cytosol, and extracellular space) [6]. The occurrence of NiSODs were first reported in Streptomyces species and then in cyanobacteria [7, 8]. MnSODs are appeared to be in one form and are accepted to be located in all plant genomes for protection from damages caused by ROS in mitochondria [9, 10]. Plant MnSODs are detected in both peroxisomes and mitochondria, and their quantities are not related to each other [11]. Plant Cu/ZnSODs contain a similar homologous group except cytosolic and chloroplast Cu/ZnSODs. The gene structures of cytosolic and chloroplast Cu/ZnSODs are different than the others by various intron positions and numbers. Cu/ZnSOD is a dimeric enzyme, having an active site with one copper and zinc. There is a histidine imidazole bridge between monomeric units [6]. Plant FeSODs show 70 % homology to plant MnSODs, but they are distantly related with each other [8, 12]. FeSOD in the chloroplasts could be related with the plastid nucleoid and is participated in signaling or gene regulation [13]. MnSODs and FeSODs contain dimers or tetramers with identical subunits. Each subunit consists of two domains: α-helical N-terminal domain and a mixed α/β C-terminal domain [14]. In Arabidopsis, seven SOD genes were identified: MSD1 (one MnSOD), FSD1, FSD2, and FSD3 (three FeSODs), and CSD1, CSD2, and CSD3 (three Cu/ZnSODs) [15]. Chloroplastic Cu/ZnSOD1 gene was isolated and characterized in maize. A slight homology was found between cDNAs of SOD1 (chloroplastic) and of SOD2, SOD4 and SOD4A (cytosolic) [16]. In sunflower, two new genes of mitochondrial MnSOD type were detected by utilization of stress conditions: low temperature (abiotic) and mechanical wounding to generate infection by Plasmopara halstedii (biotic stress) [17]. Similarly, FeSOD with an unusual subcellular localization was found and isolated in Vigna unguiculata (cowpea). This type has a homodimeric structure and is localized in the cytosol [18]. Brachypodium distachyon (L.) Beauv. (hereafter Brachypodium) has important potential for temperate cereals and forage grasses and its genome has been sequenced recently
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(International Brachypodium Initiative, 2010). Brachypodium includes many desirable features such as small diploid genome size (~300 Mbp), small physical stature, short life cycle, and self fertility [19, 20]. In the present study, several analyzes were performed by using bioinformatics tools on FeSODs, Cu/ZnSODs, and MnSODs of B. distachyon to understand molecular and structural differences of different SOD types. Additionally, 3D structures with predicted binding sites and conserved domain signatures of B. distachyon SODs were generated.
Materials and Methods Sequence Data Protein sequences of SODs from B. distachyon were downloaded from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/). A total of six SOD protein sequences (one MnSOD, two FeSODs, and three Cu/ZnSODs) were analyzed (Table 1). Primary and Secondary Structure Analyses Physico-chemical analyzes (number of amino acids and theoretical pI) were performed by using ProtParam tool (http://web.expasy.org/protparam/) [21]. Secondary structure prediction, conserved protein motifs, and predicted domain analyzes were carried out with SOPMA server [22], MEME (Multiple Em for Motif EliCitation) software [23] and Pfam server (http://pfam.sanger.ac.uk/), respectively. Predictions of subcellular localizations of SOD proteins were done with CELLO v.2.5 (subCELlular Localization predictor) [24] and WoLF PSORT [25] servers. Exons and intron structures of SOD genes from B. distachyon were identified by utilizing GSDS (Gene Structure Display Server) (http:// gsds.cbi.pku.edu.cn/) [26]. Prediction of 3D Structures and Model Evaluation The predicted 3D structures of SODs of B. distachyon were generated by using 3D Ligand Site server (http://www.sbg.bio.ic.ac.uk/3dligandsite/) [27]. Also, predicted binding sites and heterogens presences in predicted binding sites were analyzed with this server. Domain signatures of SOD proteins were predicted by using Swiss-Pdb Viewer [28]. Structural evaluation and stereo-chemical analyzes were performed by utilization of Rampage Ramachandran plot analysis (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) [29]. Phylogenetic Analysis A total of 18 SOD protein sequences, including B. distachyon (6), Arabidopsis thaliana (3), Triticum aestivum (3), Zea mays (3), and Oryza sativa (3) were used for phylogenetic analysis. These sequences were aligned by using the ClustalW program (http://www2.ebi.ac.uk/ clustalw) [30]. Evaluation for phylogenetic analysis was done by bootstrap analyzes with 1000 replications [31] with MEGA 5.1 software [32]. The phylogenetic relationship was inferred by using the Neighbor-Joining method based on the JTT (Jones–Taylor–Thomton) matrix-based model [33]. The evolutionary distances were computed with the Poisson correction method [34].
XP_003568674 XP_003561161
XP_003557205
XP_003574947
XP_003562484
XP_003558478
B. distachyon B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
Superoxide dismutase [Cu/Zn] 2-like
Superoxide dismutase [Cu/Zn] 4A-like
Chloroplastic-like SOD
Chloroplastic-like SOD [Fe]
Mitochondrial-like SOD [Mn] Chloroplastic-like SOD [Fe]
Protein ID
164
152
204
256
230 398
Number of amino acids
6.38
5.61
5.79
8.65
7.11 9.19
Theoretical pI
CZ
CZ
CZ
IM
IM IM
Predicted Pfam domain
Cy
Cy
Ch
Ch
M Ch
Subcellular prediction by CELLO
IM iron/manganese superoxide dismutase domain, CZ copper/zinc superoxide dismutase domain, M mitochondrion, Ch chloroplast, Cy cytoplasm
NCBI accession number
Species
Table 1 Physico-chemical and biochemical properties of SOD proteins in B. distachyon
Cy
Cy
Ch
Ch
M Ch
Subcellular prediction by PSORT
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Results and Discussion Sequence Analysis In the current study, a total of six SOD proteins of B. distachyon were analyzed and modeled by using bioinformatics tools. Physico-chemical analysis showed that Mn/FeSODs and Cu/ ZnSODs of B. distachyon were found to be in basic and acidic character, respectively (Table 1). Based on domain analysis, Fe/MnSODs and Cu/ZnSODs consist of iron/manganese SOD and copper–zinc SOD domains, respectively (Table 1). The predictions of subcellular localizations of SODs from B. distachyon were indicated that they were located in mitochondrion (MnSOD), chloroplast (FeSODs and one type of Cu/ZnSOD), and cytosol (two types of Cu/ZnSODs). MnSOD was detected in several prokaryotes and eukaryotes. In eukaryotes, MnSOD was observed in the matrix of the mitochondria [35]. FeSODs were frequently located in plastids and occurrence of Cu/ZnSODs was showed in plant cytosol, peroxisomes and plastids [15]. There is an agreement between the given information and our findings. Dehury et al. [36] reported that Cu/ZnSOD and MnSOD were found to be in acidic character whereas FeSOD was found to be in basic character in rice. A similarity was noticed between our results and the previous study done by Dehury et al. [36] showing Cu/ZnSODs and FeSODs of rice and B. distachyon were having acidic and basic characters, respectively. Based on the conserved motif analysis, three conserved motifs were observed: motif I — GLHGFHIHAFGDTTNGCMSTGPHFNPNGKTHGAPEDEVRHAGDLGNI, motif II — QIPLSGPHSIIGRAVVVHEDPDDLGKGGHELSKSTGNAGARIACGIIGLQ, and motif III — PLLAIDVWEHAYYLDYKNDRPDYVSNIW (Fig. 1). The motif III was found to be in FeSODs and MnSOD while motif I and II were observed in Cu/ ZnSODs. Pfam analyzes reveal that motif I and II have the copper/zinc superoxide dismutase (SODC) domain whereas motif I has the iron/manganese SOD domain and this data from Pfam analyzes support our results. Gene structure analysis for SOD genes of B. distachyon revealed that the numbers and positions of introns were varied between 5 and 7 (Fig. 2). MnSOD (Fig. 2a), FeSODs (Fig. 2b and c), and Cu/ZnSODs (Fig. 2d, e, and f) consisted of five, seven, six, seven, six and six introns, respectively. Remarkably, MnSOD had only five introns except from the other SOD genes (Fig. 2a). It was noticed that the intron numbers were different in chloroplastic (7) and cytosolic (6) Cu/ZnSODs. Fink and Scandalios (2002) reported that all cytosolic and chloroplastic SODs consisted of seven introns except one having eight introns [12]. These results show no similarity with our findings. There were only two SODs, one from FeSODs (Fig. 2b) and one from Cu/ZnSODs (Fig. 2d) having seven introns among a total of five chloroplastic and cytosolic SODs. Cu/ZnSODs containing different the number and position of introns showed no exon–intron structure similarities in related species [12]. Cu/ZnSODs of B. distachyon had the various intron patterns; thus, there was a consistency between our findings and the data from previous studies. The sizes and numbers of introns in genes are vary depending on gene and organism types and may be it is related with functional constraint on introns [37]. The differences of intron patterns in B. distachyon could be explained by the functional constraint on introns of SOD genes. In addition, it is possible that intron losses may occur in SOD genes during the genome evolution of B. distachyon. Secondary Structure Analysis The secondary structures of the SOD proteins were predicted by using SOPMA server (Table 2). The percentages of alpha helixes in MnSOD and FeSODs were
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Fig. 1 a The combined block diagram of conserved protein motifs in SODs of B. distachyon (Each motif is represented in boxes with different colors: motif 1, cyan; motif 2, blue; motif 3, red.) b The conserved motif logos (motif I, motif II, and motif III, respectively)
higher than Cu/ZnSODs. Chloroplastic, cytosolic, and peroxisomal Cu/ZnSODs of soybean and Arabidopsis had no helix structures [38]. This data show similarity with ours. Also, Cu/ZnSODs of B. distachyon had no helices. However, Mn/FeSODs of B. distachyon consisted of more helices, suggesting the quality index. Notably, random coils generally were in higher rates in all SODs of B. distachyon. Random coils play important roles in proteins related with their flexibility and conformational changes, including enzymatic turnover [39]. Our data suggest that the random coils of SODs in B. distachyon play roles related with the conformational changes of SOD proteins. Predicted binding sites of SODs of B. distachyon were different in Fe/MnSODs and Cu/ZnSODs (Table 3). The predicted binding sites of Fe/MnSODs and Cu/ ZnSODs were having His-His-Asp-His and His-His-His-Asp-Ser residues, respectively with different positions. In rice, His-His-Asp-His residues in Mn/FeSODs, His-HisHis-His residues in CuSOD and His-His-His-Asp residues in ZnSOD were observed as predicted binding sites, respectively [36]. These findings are in agreement with our results. In the active site of Cu/ZnSODs, one Cu ion and one Zn ion are ligated by three histidines, one aspartic acid, and three histidines, respectively [35]. The active site of FeSODs has a single Fe ion coordinated in a trigonal bipyramid by three histidines and an Asp [40]. Our findings are consistent with the data done previously. Cu/ZnSODs and Fe/MnSODs consisted of same residues with different positions in predicted binding sites. The heterogens present in SOD proteins of B. distachyon were similar to each other: Zn for Cu/ZnSODs and Fe and Fe+2 for Mn/FeSODs (Table 4).
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Fig. 2 Structure of SOD genes from B. distachyon. Exons and introns are depicted by filled green boxes and single lines, respectively. Intron phases 0, 1, and 2 are indicated by numbers 0, 1, and 2. Untranslated regions (UTRs) are displayed by thick blue lines at both 5′ and 3′ ends of each gene. (a XP_003568674, b XP_003561161, c XP_003557205, d XP_003574947, e XP_003562484, f XP_003558478)
Predicted 3D Structures and Binding Sites Protein three-dimensional structure is more conserved than protein sequences or functions and being shaped with a combination of mechanisms such as gene duplication, mutation, and selection. Also, the modifications such as insertion/deletion/substitution of secondary structural elements, circular permutation, β-strand invasion/withdrawal, and β-hairpin flip/swap on protein folding are main factors in domain evolution [41, 42]. Here, the predicted 3D structures and binding sites of SOD proteins of B. distachyon were generated by using 3D LigandSite server and predicted binding residues were resembled by blue color in 3D structures (Fig. 3). Based on 3D data, helical structures were detected in the types of Mn/FeSODs in contrary to Cu/ZnSODs having no helical structures. Gopavajhula et al. [37] reported that there were no helical structures in Cu/ZnSODs of soybean and Arabidopsis and the data from previous studies and ours show similarity (Fig. 3d, e, and f) [38]. In rice, the strands were observed as dominant secondary structures in Cu/ZnSODs whereas helical structures were found to be dominant in Fe/MnSODs [36]. These findings corroborate our data. Also, it was noticed that Table 2 Secondary structure analysis of Brachypodium SODs by using SOPMA server Secondary structure
XP_003568674 XP_003561161 XP_003557205 XP_003574947 XP_003562484 XP_003558478 MnSOD FeSOD FeSOD Cu/ZnSOD Cu/ZnSOD Cu/ZnSOD
Alpha helix (Hh)
50.00 %
37.19 %
39.84 %
19.12 %
5.26 %
10.37 %
Extended strand (Ee)
13.00 %
14.57 %
15.23 %
29.90 %
36.18 %
30.49 %
Beta turn (Tt)
6.09 %
4.77 %
3.91 %
5.39 %
8.55 %
10.37 %
43.47 %
41.02 %
45.59 %
50.00 %
48.78 %
Random coil (Cc) 30.87 %
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Table 3 Predicted binding sites of SOD proteins of B. distachyon
Protein
Accession no.
Mn SOD
XP_003568674 54
Fe SOD
Residue Amino Contact Ave. acid distance His
23
0.53
102
His
25
0.52
191
Asp
25
0.52
195
His
25
0.46
XP_003561161 145
His
17
0.67
199 298
His Asp
24 24
0.20 0.29 0.49
302
His
24
His
24
0.48
120
His
24
0.45
204
Asp
24
0.23
208
His
24
0.33
Cu/Zn SOD XP_003574947 113
His
25
0.00
121 130
His His
25 25
0.00 0.00
133
Asp
25
0.00
186
Ser
23
0.58
Cu/Zn SOD XP_003562484 62
His
25
0.00
70
His
25
0.00
79
His
25
0.00
82
Asp
25
0.00
Ser His
25 25
0.47 0.00
80
His
25
0.00
89
His
25
0.00
92
Asp
25
0.00
145
Ser
22
0.58
Fe SOD
XP_003557205 68
165 Cu/Zn SOD XP_003558478 72
chloroplastic (Fig. 3d) and cytosolic Cu/ZnSODs (Fig. 3f) were identical. It can be stated that Cu/ZnSODs may evolve from the same ancestral SOD genes during genome evolution of B. distachyon. Fe/MnSODs of B. distachyon had helices and three β-sheets (Fig. 3a, b, and c), Table 4 Heterogens present in predicted binding sites
Protein
Accession no.
Mn SOD
XP_003568674
Fe SOD Fe SOD
XP_003561161 XP_003557205
Heterogen
Count
Fe
22
Fe2
3
Fe
19
Fe2
5
Fe
19
Fe2
5
Cu/Zn SOD Cu/Zn SOD
XP_003574947 XP_003562484
Zn Zn
25 25
Cu/Zn SOD
XP_003558478
Zn
25
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while Cu/ZnSODs of B. distachyon had nine β-sheets (Fig. 3d, e, and f) and disulfide bridges (Cys107/Cys196 in XP_003574947 (Fig. 3d); Cys56/Cys145 in XP_003562484 (Fig. 3e), and Cys66/Cys155 in XP_003558478 (Fig. 3f), showing the stability of protein folds as reported in rice [36]. Cu/ZnSODs of eukaryotic organisms consist of highly conserved structures and are composed of two identical subunits. Each subunit includes a β-barrel with eight antiparallel β-strands [42]. The Cu and Zn sites are located at the outside of the β-barrel in the active site channel [35]. In SODs of B. distachyon, β-barrel structures were observed and metal binding sites were located at the outside of the β-barrel structures (Fig. 3d, e, and f) as well, and these are in agreement with the previous data. In Mn/FeSODs, the polypeptide chain contains Nterminal helices and a C-terminal α/β domain. The active sites of Mn/FeSODs are positioned between the N- and C-terminal domains [35, 43]. These data support our findings indicating Mn/FeSODs of B. distachyon had the helix bundles and similar domain structures (Fig. 3a, b and c). The well-defined signature sequences are used for establishing evolutionary relationships among species [44]. Although their locations were different (Fig. 4a and b), conserved signatures (highly conserved regions) were observed in both cytocolic and chloroplastic Cu/ ZnSODs based on the domain analysis of Cu/ZnSODs in B. distachyon. Some residues, including Gly91, Leu92, His93, Gly94, Phe95, His96, Leu97, His98, and Glu99 were observed in chloroplastic Cu/ZnSODs (Fig. 4a), while some residues, including Gly40, Leu41, His42, Gly43, Phe44, His45, Val46, His47, and Ala48 were seen in cytocolic Cu/ ZnSODs (Fig. 4b) of conserved pattern of copper/zinc SOD domains. These residues contribute to β-sheet formation of Cu/ZnSODs in B. distachyon. It can be said that 3D structures of
Fig. 3 Prediction of 3D structures and binding sites of SODs in B. distachyon done by using the 3D LigandSite server (a XP_003568674, MnSOD mitochondrial; b XP_003561161, FeSOD chloroplast; c XP_003557205, FeSOD chloroplast; d XP_003574947, Cu/ZnSOD chloroplast; e XP_003562484, Cu/ZnSOD cytosol; f XP_003558478, Cu/ZnSOD cytosol)
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Fig. 4 Solid ribbon representations of chloroplastic Cu/ZnSOD (XP_003574947) (a) and cytosolic Cu/ZnSOD (XP_003562484) (b) for Pfam patterns of copper/zinc superoxide dismutase domain and mitochondrial MnSOD (XP_003568674) (c) and chloroplastic FeSOD (XP_003561161) (d) for Pfam patterns of iron/manganese superoxide dismutase domain. The structural divergences are indicated as blue empty circles
Cu/ZnSODs in B. distachyon showed structural divergence in domain patterns (Fig. 4a and b). Thus, these divergences could be related with the differences in gene structures of chloroplastic and cytocolic SODs of B. distachyon [12]. Some identified residues such as His195, Ala196, Tyr197, Tyr198, Leu199, Gln200 and Tyr201 in MnSOD of B. distachyon as shown in Fig. 3c and His302, Ala303, Tyr304, Tyr305, Leu306, Asp307 and Tyr308 in FeSOD of B. distachyon as seen in Fig. 3d contribute for the construction of helix structures in B. distachyon. Furthermore, additional β-sheet structure was available in MnSOD of B. distachyon (Fig. 4c) posing effects on the enzyme–substrate interactions and the activity of enzymes in stress conditions. Although, the most of these residues were in same sequences for Cu/ZnSODs (Gly, Leu, His, Gly, Phe, His, X, His, and Y) and Fe/MnSODs (His, Ala, Tyr,
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Fig. 5 Ramachandran plot analyzes of SODs in B. distachyon by using Rampage server (a XP_003568674, MnSOD; b XP_003561161, FeSOD; c XP_003557205, FeSOD; d XP_003574947, Cu/ZnSOD; e XP_003562484, Cu/ZnSOD; f XP_003558478, Cu/ZnSOD)
Tyr, Leu, Z and Tyr), substitutions were occurred in some residues of Cu/ZnSODs (Leu/Val and Glu/Ala) and Fe/MnSODs (Gln/Asp) in B. distachyon. These findings may be related with some genome dynamics, including insertion, deletion, and substitutions in the SOD genes of in B. distachyon. Model Validation For model validation, the Ramachandran plot analyses were performed by utilization of the RAMPAGE server. According to the results, 92.5 %, 93.4 %, 94.1 %, 85.5 %, 94.7 %, and 87.7 % were in favored region, 4 %, 5.1 %, 3.9 %, 9.2 %, 5.3 % and 7.1 % in allowed region and 3.5 %, 1.5 %, 2 %, 5.3 %, 0 % and 5.2 %% in outlier region in MnSOD (XP_003568674), FeSOD (XP_003561161), FeSOD (XP_003557205), Cu/ZnSOD (XP_003574947), Cu/ ZnSOD (XP_003562484), and Cu/ZnSOD (XP_003558478) of B. distachyon, respectively (Fig. 5), suggesting that the 3D models were fairly in good quality. Phylogenetic Analysis In order to understand the phylogenetic relationships of B. distachyon SODs; SOD protein sequences of some grass species (rice, maize, wheat, and Brachypodium) and Arabidopsis
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Fig. 6 Phylogenetic tree of SOD protein sequences in higher plant species, including B. distachyon, A. thaliana, T. aestivum, Z. mays, and O. sativa. Sequence alignment was performed by using ClustalX and phylogenetic tree drawn by Neighbor-Joining method with MEGA 5.1. SODs of B. distachyon are shown in red boxes
were used. The phylogenetic analyzes revealed that plant SODs were separated into two main groups: Mn/FeSOD and Cu/ZnSOD (Fig. 6). As in Brachypodium, Mn/FeSODs and Cu/ ZnSODs were found to be separately clustered in soybean [37] and SODs of rice [36, 45]. Plant MnSODs exhibiting 70 % homology are more distantly related to plant FeSODs indicating separate evolutionary origins [12]. Our results could be related with the separate evolutionary origins of Mn/FeSODs. Mn/FeSOD group was separated into two subgroups: FeSODs and MnSODs. Also, Cu/ZnSOD group was divided into two subgroups: chloroplastic and cytosolic Cu/ZnSODs. In our phylogenetic tree, Mn/FeSODs were grouped together, consisting of both monocots and dicots. Interestingly, MnSODs (dicot) of Arabidopsis were clustered in MnSODs of monocots with the highest bootstrap value (100 %). However, FeSODs of Arabidopsis and Brachypodium were grouped together with 77 % of bootstrap value. It can be proposed that the genes of MnSODs are well conserved and may have been evolved from a common ancestral gene in monocots and dicots. In Cu/ZnSODs, chloroplast Cu/ZnSODs were grouped together with a high bootstrap value (98 %) whereas the cytosolic Cu/ZnSODs were grouped together with a low bootstrap value (64 %). The chloroplastic Cu/ZnSOD (XP_003574947) of B. distachyon and Triticum Cu/ ZnSOD were grouped together with 82 % of bootstrap value. Plant Cu/ZnSODs observed in cytosols, chloroplasts and also peroxisomes; contain a highly homologous group. However, cytosolic and chloroplastic Cu/ZnSODs have the distinctive features with the numbers and positions of introns [12]. This information supports our findings showing the separation of chloroplastic Cu/ZnSODs and cytosolic Cu/ZnSODs. This separation may be affected from gene structure, consisting various numbers and positions of introns (Fig. 2). In conclusion, the molecular structures of SODs are important for understanding of response to oxidative stress in plants. Thus, sequence analyzes and structural models of Mn, Fe and Cu/ZnSODs in B. distachyon were evaluated by employing bioinformatics tools. These analyzes provide insights for molecular functions of SOD isoenzymes related with metal bindings in different cellular organelles. Secondary structural analyzes and predicted binding
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sites of Cu/ZnSODs and Mn/FeSODs of B. distachyon were well conserved, whereas some domain signatures were structurally showed divergences. These binding sites could be important for analyzes of functional site residues in SODs during catalytic processes. Phylogenetic analyzes showed that Fe/MnSODs and Cu/ZnSODs in B. distachyon were distributed separately emphasizing originations of Fe/MnSODs and Cu/ZnSODs from different ancestors. Also, results of this study may contribute to in silico comparative studies about SODs in plants, particularly in grasses.
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Uzilday, B., Turkan, I., Ozgur, R., & Sekmen, A. H. (2014). Journal of Plant Physiology, 171, 65–75. Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Journal of Botany. doi:10.1155/2012/217037. Smirnoff, N. (1993). New Phytologist, 125, 27–58. Sainz, M., Diaz, P., Monza, J., & Borsani, O. (2010). Physiologia Plantarum, 140, 46–56. Liochev, S., & Fridovich, I. (1994). Free Radical Biology and Medicine, 16, 29–33. Abreu, I. A., & Cabelli, D. E. (2010). Biochimica et Biophysica Acta, 1804, 263–274. Youn, H. D., Kim, E. J., Roe, J. H., Hah, Y. C., & Kang, S. O. (1996). Biochemical Journal, 318, 889–896. Miller, A. F. (2012). FEBS Letters, 586, 585–595. Carlioz, A., & Touati, D. (1986). EMBO Journal, 5, 623–630. Møller, I. M. (2001). Annual Review of Plant Physiology and Plant Molecular Biology, 52, 561–591. del Río, L. A., Sandalio, L. M., Altomare, D. A., & Zilinskas, B. A. (2003). Journal of Experimental Botany, 54, 923–933. Fink, R. C., & Scandalios, J. G. (2002). Archives of Biochemistry and Biophysics, 399, 19–36. Pilon, M., Ravet, K., & Tapken, W. (2011). Biochimica et Biophysica Acta, 1807, 989–998. Whittaker, J. W. (2003). Biochemical Society Transactions, 31, 1318–1321. Kliebenstein, D. J., Monde, R. A., & Last, R. L. (1998). Plant Physiology, 118, 637–650. Kernodle, S. P., & Scandalios, J. G. (2001). Archives of Biochemistry and Biophysics, 391, 137– 147. Fernández-Ocána, A., Chaki, M., Luque, F., Gómez-Rodríguez, M. V., Carreras, A., Valderrama, R., et al. (2011). Journal of Plant Physiology, 168, 1303–1308. Muñoz, I. G., Moran, J. F., Becana, M., & Montoya, G. (2005). Protein Science, 14, 387–394. Draper, J., Mur, L. A. J., Jenkins, G., Ghosh-Biswas, G. C., Bablak, P., Hasterok, R., et al. (2001). Plant Physiology, 127, 1539–1555. Ozdemir, B. S., Hernandez, P., Filiz, E., & Budak, H. (2008). International Journal of Plant Genomics. doi: 10.1155/2008/536104. Article ID 536104. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., et al. (2005). In J. M. Walker (Ed.), The proteomics protocols handbook (pp. 571–607). Totowa, NJ, USA: Humana Press. Geourjon, C., & Deléage, G. (1995). Computer Applications in the Biosciences, 11, 681–684. Timothy, L., Mikael Bodén, B., Buske, F. A., Frith, M., Grant, C. E., Clementi, L., et al. (2009). Nucleic Acids Research, 37, 202–208. Yu, C. S., Chen, Y. C., Lu, C. H., & Hwang, K. (2006). Proteins: Structure, Function, and Bioinformatics, 64, 643–651. Horton, P., Park, K., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., et al. (2007). Nucleic Acids Research. doi:10.1093/nar/gkm259. Guo, A. Y., Zhu, Q. H., Chen, X., & Luo, J. C. (2007). Yi Chuan, 29, 1023–1026. Wass, M. N., Kelley, L. A., & Sternberg, M. J. (2010). Nucleic Acids Research, 38(Suppl), 469–473. Guex, N., Peitsch, M. C., & Schwede, T. (2009). Electrophoresis, 1(Suppl), S162–S173. Lovell, S. C., Davis, I. W., Arendall, W., Bakker, P. I. W., Word, J. M., Prisant, M. G., et al. (2003). Proteins: Structure, Function, and Genetics, 50, 437–450. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., et al. (2007). Bioinformatics, 23, 2947–2948. Felsenstein, J. (1985). Evolution, 39, 783–791. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). Molecular Biology and Evolution, 28, 2731–2739. Jones, D. T., Taylor, W. R., & Thornton, J. M. (1992). Computer Applications in the Biosciences, 8, 275– 282.
Appl Biochem Biotechnol 34. Zuckerkandl, E., & Pauling, L. (1965). Edited in Evolving Genes and Proteins by Bryson V. Vogel HJ: Academic Press New York. 35. Perry, J. J. P., Shin, D. S., Getzoff, E. D., & Tainer, J. A. (2010). Biochimica et Biophysica Acta, 1804, 245– 262. 36. Dehury, B., Sarma, K., Sarmah, R., Sahu, J., Sahoo, S., Sahu, M., et al. (2012). Journal of Plant Biochemistry and Biotechnology, DOI 10.1007/s13562-012-0121-6. Long M 2002. 37. Gopavajhula, V. R., Chaitanya, K. V., Khan, P. A. A., Shaik, J. P., Reddy, P. N., & Alanazi, M. (2013). Genetics and Molecular Biology, 36, 225–236. 38. Buxbaum, E. (2007). Fundamentals of protein structure and function. New York: Springer Science Business Media. 39. Miller, A. F. (2004). Current Opinion in Chemical Biology, 8, 162–168. 40. Grishin, N. V. (2001). Journal of Structural Biology, 134, 167–185. 41. Kinch, L. N., & Grishin, N. V. (2002). Current Opinion in Structural Biology, 12, 400–408. 42. Richardson, J. S. (1977). Nature, 268, 495–500. 43. Lah, M. S., Dixon, M. M., Pattridge, K. A., Stallings, W. C., Fee, J. A., & Ludwig, M. L. (1995). Biochemistry, 34, 1646–1660. 44. Gupta, R. S. (1998). Microbiology and Molecular Biology Reviews, 62, 1435–1491. 45. Balasubramanian, A., Das, S., Bora, A., Sarangi, S., & Mandal, A. B. (2012). American Journal of Plant Sciences, 3, 1311–1321.
Comparative Analysis and Modeling of Superoxide Dismutases (SODs) in Brachypodium distachyon L. Ertugrul Filiz & Ibrahim Koc & Ibrahim Ilker Ozyigit
Received: 21 January 2014 / Accepted: 14 April 2014 # Springer Science+Business Media New York 2014
Abstract Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of superoxide radical to hydrogen peroxide and dioxygen. To date, four types of SODs — Cu/ZnSOD, MnSOD, FeSOD, and NiSOD — have been identified. In this study, SOD proteins of Brachypodium distachyon (L.) Beauv. were screened by utilization of bioinformatics approaches. According to our results, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found to be in basic and acidic character, respectively. Domain analyzes of SOD proteins revealed that iron/manganese SOD and copper/zinc SOD were within studied SOD proteins. Based on the seconder structure analyzes, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found as having similar sheets, turns and coils. Although helical structures were noticed in the types of Mn/ FeSODs, no the type of Cu/ZnSODs were identified having helical structures. The predicted binding sites of Fe/MnSODs and Cu/ZnSODs were confirmed for having His-His-Asp-His and His-His-His-Asp-Ser residues with different positions, respectively. The 3D structure analyzes of SODs revealed that some structural divergences were observed in patterns of SODs domains. Based on phylogenetic analysis, Mn/FeSODs were found to have similarities whereas Cu/ZnSODs were clustered independently in phylogenetic tree. Keywords Superoxide dismutase . Antioxidant proteins . Brachypodium distachyon . 3D modeling . In silico analysis
E. Filiz (*) Department of Crop and Animal Production, Cilimli Vocational School, Duzce University, 81750 Cilimli, Duzce, Turkey e-mail: [email protected] I. Koc Faculty of Science, Department of Molecular Biology and Genetics, Gebze Institute of Technology, Gebze, Kocaeli 41400, Turkey I. I. Ozyigit Faculty of Science and Arts, Department of Biology, Marmara University, 34722 Goztepe, Istanbul, Turkey
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Abbreviations SOD Superoxide dismutase FeSOD Iron superoxide dismutase MnSOD Manganese superoxide dismutase Cu/ZnSOD Copper/zinc superoxide dismutase ROS Reactive oxygen species Introduction Reactive oxygen species (ROS) are free radicals derived from O2 and are stimulated in various types of biotic and abiotic stresses. They can pose damages to biomolecules such as membrane and proteins at high concentrations whereas they can show functions as second messengers at low/moderate concentrations [1]. The most common types of ROS are hydrogen peroxide (H2O2), hypochlorite (OCl−), peroxynitrate (ONO2−), and hydroxyl radical (HO.), which can be removed by antioxidants and antioxidative enzymes. Major ROS-scavenging enzymes in plants are superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PrxR). SODs (EC 1.15.1.1) and CAT (EC 1.11.1.6) as ROS-scavenging systems save plant cells by decreasing the toxic forms of oxygen [2–4]. SODs (EC 1.15.1.1) are members of metalloenzymes catalyzing the dismutation of superoxide to oxygen and hydrogen peroxide [5]. Based on prosthetic metals, SODs can be categorized in four (canonical) groups: iron SOD (FeSOD), manganese SOD (MnSOD), copper/zinc SOD (Cu/ZnSOD), and nickel SOD (NiSOD). Although all types of SODs are found in prokaryotic organisms, eukaryotes only have three types: FeSODs (in chloroplasts), MnSODs (in mitochondria and peroxisomes), and Cu/ZnSODs (in chloroplast, cytosol, and extracellular space) [6]. The occurrence of NiSODs were first reported in Streptomyces species and then in cyanobacteria [7, 8]. MnSODs are appeared to be in one form and are accepted to be located in all plant genomes for protection from damages caused by ROS in mitochondria [9, 10]. Plant MnSODs are detected in both peroxisomes and mitochondria, and their quantities are not related to each other [11]. Plant Cu/ZnSODs contain a similar homologous group except cytosolic and chloroplast Cu/ZnSODs. The gene structures of cytosolic and chloroplast Cu/ZnSODs are different than the others by various intron positions and numbers. Cu/ZnSOD is a dimeric enzyme, having an active site with one copper and zinc. There is a histidine imidazole bridge between monomeric units [6]. Plant FeSODs show 70 % homology to plant MnSODs, but they are distantly related with each other [8, 12]. FeSOD in the chloroplasts could be related with the plastid nucleoid and is participated in signaling or gene regulation [13]. MnSODs and FeSODs contain dimers or tetramers with identical subunits. Each subunit consists of two domains: α-helical N-terminal domain and a mixed α/β C-terminal domain [14]. In Arabidopsis, seven SOD genes were identified: MSD1 (one MnSOD), FSD1, FSD2, and FSD3 (three FeSODs), and CSD1, CSD2, and CSD3 (three Cu/ZnSODs) [15]. Chloroplastic Cu/ZnSOD1 gene was isolated and characterized in maize. A slight homology was found between cDNAs of SOD1 (chloroplastic) and of SOD2, SOD4 and SOD4A (cytosolic) [16]. In sunflower, two new genes of mitochondrial MnSOD type were detected by utilization of stress conditions: low temperature (abiotic) and mechanical wounding to generate infection by Plasmopara halstedii (biotic stress) [17]. Similarly, FeSOD with an unusual subcellular localization was found and isolated in Vigna unguiculata (cowpea). This type has a homodimeric structure and is localized in the cytosol [18]. Brachypodium distachyon (L.) Beauv. (hereafter Brachypodium) has important potential for temperate cereals and forage grasses and its genome has been sequenced recently
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(International Brachypodium Initiative, 2010). Brachypodium includes many desirable features such as small diploid genome size (~300 Mbp), small physical stature, short life cycle, and self fertility [19, 20]. In the present study, several analyzes were performed by using bioinformatics tools on FeSODs, Cu/ZnSODs, and MnSODs of B. distachyon to understand molecular and structural differences of different SOD types. Additionally, 3D structures with predicted binding sites and conserved domain signatures of B. distachyon SODs were generated.
Materials and Methods Sequence Data Protein sequences of SODs from B. distachyon were downloaded from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/). A total of six SOD protein sequences (one MnSOD, two FeSODs, and three Cu/ZnSODs) were analyzed (Table 1). Primary and Secondary Structure Analyses Physico-chemical analyzes (number of amino acids and theoretical pI) were performed by using ProtParam tool (http://web.expasy.org/protparam/) [21]. Secondary structure prediction, conserved protein motifs, and predicted domain analyzes were carried out with SOPMA server [22], MEME (Multiple Em for Motif EliCitation) software [23] and Pfam server (http://pfam.sanger.ac.uk/), respectively. Predictions of subcellular localizations of SOD proteins were done with CELLO v.2.5 (subCELlular Localization predictor) [24] and WoLF PSORT [25] servers. Exons and intron structures of SOD genes from B. distachyon were identified by utilizing GSDS (Gene Structure Display Server) (http:// gsds.cbi.pku.edu.cn/) [26]. Prediction of 3D Structures and Model Evaluation The predicted 3D structures of SODs of B. distachyon were generated by using 3D Ligand Site server (http://www.sbg.bio.ic.ac.uk/3dligandsite/) [27]. Also, predicted binding sites and heterogens presences in predicted binding sites were analyzed with this server. Domain signatures of SOD proteins were predicted by using Swiss-Pdb Viewer [28]. Structural evaluation and stereo-chemical analyzes were performed by utilization of Rampage Ramachandran plot analysis (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) [29]. Phylogenetic Analysis A total of 18 SOD protein sequences, including B. distachyon (6), Arabidopsis thaliana (3), Triticum aestivum (3), Zea mays (3), and Oryza sativa (3) were used for phylogenetic analysis. These sequences were aligned by using the ClustalW program (http://www2.ebi.ac.uk/ clustalw) [30]. Evaluation for phylogenetic analysis was done by bootstrap analyzes with 1000 replications [31] with MEGA 5.1 software [32]. The phylogenetic relationship was inferred by using the Neighbor-Joining method based on the JTT (Jones–Taylor–Thomton) matrix-based model [33]. The evolutionary distances were computed with the Poisson correction method [34].
XP_003568674 XP_003561161
XP_003557205
XP_003574947
XP_003562484
XP_003558478
B. distachyon B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
Superoxide dismutase [Cu/Zn] 2-like
Superoxide dismutase [Cu/Zn] 4A-like
Chloroplastic-like SOD
Chloroplastic-like SOD [Fe]
Mitochondrial-like SOD [Mn] Chloroplastic-like SOD [Fe]
Protein ID
164
152
204
256
230 398
Number of amino acids
6.38
5.61
5.79
8.65
7.11 9.19
Theoretical pI
CZ
CZ
CZ
IM
IM IM
Predicted Pfam domain
Cy
Cy
Ch
Ch
M Ch
Subcellular prediction by CELLO
IM iron/manganese superoxide dismutase domain, CZ copper/zinc superoxide dismutase domain, M mitochondrion, Ch chloroplast, Cy cytoplasm
NCBI accession number
Species
Table 1 Physico-chemical and biochemical properties of SOD proteins in B. distachyon
Cy
Cy
Ch
Ch
M Ch
Subcellular prediction by PSORT
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Results and Discussion Sequence Analysis In the current study, a total of six SOD proteins of B. distachyon were analyzed and modeled by using bioinformatics tools. Physico-chemical analysis showed that Mn/FeSODs and Cu/ ZnSODs of B. distachyon were found to be in basic and acidic character, respectively (Table 1). Based on domain analysis, Fe/MnSODs and Cu/ZnSODs consist of iron/manganese SOD and copper–zinc SOD domains, respectively (Table 1). The predictions of subcellular localizations of SODs from B. distachyon were indicated that they were located in mitochondrion (MnSOD), chloroplast (FeSODs and one type of Cu/ZnSOD), and cytosol (two types of Cu/ZnSODs). MnSOD was detected in several prokaryotes and eukaryotes. In eukaryotes, MnSOD was observed in the matrix of the mitochondria [35]. FeSODs were frequently located in plastids and occurrence of Cu/ZnSODs was showed in plant cytosol, peroxisomes and plastids [15]. There is an agreement between the given information and our findings. Dehury et al. [36] reported that Cu/ZnSOD and MnSOD were found to be in acidic character whereas FeSOD was found to be in basic character in rice. A similarity was noticed between our results and the previous study done by Dehury et al. [36] showing Cu/ZnSODs and FeSODs of rice and B. distachyon were having acidic and basic characters, respectively. Based on the conserved motif analysis, three conserved motifs were observed: motif I — GLHGFHIHAFGDTTNGCMSTGPHFNPNGKTHGAPEDEVRHAGDLGNI, motif II — QIPLSGPHSIIGRAVVVHEDPDDLGKGGHELSKSTGNAGARIACGIIGLQ, and motif III — PLLAIDVWEHAYYLDYKNDRPDYVSNIW (Fig. 1). The motif III was found to be in FeSODs and MnSOD while motif I and II were observed in Cu/ ZnSODs. Pfam analyzes reveal that motif I and II have the copper/zinc superoxide dismutase (SODC) domain whereas motif I has the iron/manganese SOD domain and this data from Pfam analyzes support our results. Gene structure analysis for SOD genes of B. distachyon revealed that the numbers and positions of introns were varied between 5 and 7 (Fig. 2). MnSOD (Fig. 2a), FeSODs (Fig. 2b and c), and Cu/ZnSODs (Fig. 2d, e, and f) consisted of five, seven, six, seven, six and six introns, respectively. Remarkably, MnSOD had only five introns except from the other SOD genes (Fig. 2a). It was noticed that the intron numbers were different in chloroplastic (7) and cytosolic (6) Cu/ZnSODs. Fink and Scandalios (2002) reported that all cytosolic and chloroplastic SODs consisted of seven introns except one having eight introns [12]. These results show no similarity with our findings. There were only two SODs, one from FeSODs (Fig. 2b) and one from Cu/ZnSODs (Fig. 2d) having seven introns among a total of five chloroplastic and cytosolic SODs. Cu/ZnSODs containing different the number and position of introns showed no exon–intron structure similarities in related species [12]. Cu/ZnSODs of B. distachyon had the various intron patterns; thus, there was a consistency between our findings and the data from previous studies. The sizes and numbers of introns in genes are vary depending on gene and organism types and may be it is related with functional constraint on introns [37]. The differences of intron patterns in B. distachyon could be explained by the functional constraint on introns of SOD genes. In addition, it is possible that intron losses may occur in SOD genes during the genome evolution of B. distachyon. Secondary Structure Analysis The secondary structures of the SOD proteins were predicted by using SOPMA server (Table 2). The percentages of alpha helixes in MnSOD and FeSODs were
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Fig. 1 a The combined block diagram of conserved protein motifs in SODs of B. distachyon (Each motif is represented in boxes with different colors: motif 1, cyan; motif 2, blue; motif 3, red.) b The conserved motif logos (motif I, motif II, and motif III, respectively)
higher than Cu/ZnSODs. Chloroplastic, cytosolic, and peroxisomal Cu/ZnSODs of soybean and Arabidopsis had no helix structures [38]. This data show similarity with ours. Also, Cu/ZnSODs of B. distachyon had no helices. However, Mn/FeSODs of B. distachyon consisted of more helices, suggesting the quality index. Notably, random coils generally were in higher rates in all SODs of B. distachyon. Random coils play important roles in proteins related with their flexibility and conformational changes, including enzymatic turnover [39]. Our data suggest that the random coils of SODs in B. distachyon play roles related with the conformational changes of SOD proteins. Predicted binding sites of SODs of B. distachyon were different in Fe/MnSODs and Cu/ZnSODs (Table 3). The predicted binding sites of Fe/MnSODs and Cu/ ZnSODs were having His-His-Asp-His and His-His-His-Asp-Ser residues, respectively with different positions. In rice, His-His-Asp-His residues in Mn/FeSODs, His-HisHis-His residues in CuSOD and His-His-His-Asp residues in ZnSOD were observed as predicted binding sites, respectively [36]. These findings are in agreement with our results. In the active site of Cu/ZnSODs, one Cu ion and one Zn ion are ligated by three histidines, one aspartic acid, and three histidines, respectively [35]. The active site of FeSODs has a single Fe ion coordinated in a trigonal bipyramid by three histidines and an Asp [40]. Our findings are consistent with the data done previously. Cu/ZnSODs and Fe/MnSODs consisted of same residues with different positions in predicted binding sites. The heterogens present in SOD proteins of B. distachyon were similar to each other: Zn for Cu/ZnSODs and Fe and Fe+2 for Mn/FeSODs (Table 4).
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Fig. 2 Structure of SOD genes from B. distachyon. Exons and introns are depicted by filled green boxes and single lines, respectively. Intron phases 0, 1, and 2 are indicated by numbers 0, 1, and 2. Untranslated regions (UTRs) are displayed by thick blue lines at both 5′ and 3′ ends of each gene. (a XP_003568674, b XP_003561161, c XP_003557205, d XP_003574947, e XP_003562484, f XP_003558478)
Predicted 3D Structures and Binding Sites Protein three-dimensional structure is more conserved than protein sequences or functions and being shaped with a combination of mechanisms such as gene duplication, mutation, and selection. Also, the modifications such as insertion/deletion/substitution of secondary structural elements, circular permutation, β-strand invasion/withdrawal, and β-hairpin flip/swap on protein folding are main factors in domain evolution [41, 42]. Here, the predicted 3D structures and binding sites of SOD proteins of B. distachyon were generated by using 3D LigandSite server and predicted binding residues were resembled by blue color in 3D structures (Fig. 3). Based on 3D data, helical structures were detected in the types of Mn/FeSODs in contrary to Cu/ZnSODs having no helical structures. Gopavajhula et al. [37] reported that there were no helical structures in Cu/ZnSODs of soybean and Arabidopsis and the data from previous studies and ours show similarity (Fig. 3d, e, and f) [38]. In rice, the strands were observed as dominant secondary structures in Cu/ZnSODs whereas helical structures were found to be dominant in Fe/MnSODs [36]. These findings corroborate our data. Also, it was noticed that Table 2 Secondary structure analysis of Brachypodium SODs by using SOPMA server Secondary structure
XP_003568674 XP_003561161 XP_003557205 XP_003574947 XP_003562484 XP_003558478 MnSOD FeSOD FeSOD Cu/ZnSOD Cu/ZnSOD Cu/ZnSOD
Alpha helix (Hh)
50.00 %
37.19 %
39.84 %
19.12 %
5.26 %
10.37 %
Extended strand (Ee)
13.00 %
14.57 %
15.23 %
29.90 %
36.18 %
30.49 %
Beta turn (Tt)
6.09 %
4.77 %
3.91 %
5.39 %
8.55 %
10.37 %
43.47 %
41.02 %
45.59 %
50.00 %
48.78 %
Random coil (Cc) 30.87 %
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Table 3 Predicted binding sites of SOD proteins of B. distachyon
Protein
Accession no.
Mn SOD
XP_003568674 54
Fe SOD
Residue Amino Contact Ave. acid distance His
23
0.53
102
His
25
0.52
191
Asp
25
0.52
195
His
25
0.46
XP_003561161 145
His
17
0.67
199 298
His Asp
24 24
0.20 0.29 0.49
302
His
24
His
24
0.48
120
His
24
0.45
204
Asp
24
0.23
208
His
24
0.33
Cu/Zn SOD XP_003574947 113
His
25
0.00
121 130
His His
25 25
0.00 0.00
133
Asp
25
0.00
186
Ser
23
0.58
Cu/Zn SOD XP_003562484 62
His
25
0.00
70
His
25
0.00
79
His
25
0.00
82
Asp
25
0.00
Ser His
25 25
0.47 0.00
80
His
25
0.00
89
His
25
0.00
92
Asp
25
0.00
145
Ser
22
0.58
Fe SOD
XP_003557205 68
165 Cu/Zn SOD XP_003558478 72
chloroplastic (Fig. 3d) and cytosolic Cu/ZnSODs (Fig. 3f) were identical. It can be stated that Cu/ZnSODs may evolve from the same ancestral SOD genes during genome evolution of B. distachyon. Fe/MnSODs of B. distachyon had helices and three β-sheets (Fig. 3a, b, and c), Table 4 Heterogens present in predicted binding sites
Protein
Accession no.
Mn SOD
XP_003568674
Fe SOD Fe SOD
XP_003561161 XP_003557205
Heterogen
Count
Fe
22
Fe2
3
Fe
19
Fe2
5
Fe
19
Fe2
5
Cu/Zn SOD Cu/Zn SOD
XP_003574947 XP_003562484
Zn Zn
25 25
Cu/Zn SOD
XP_003558478
Zn
25
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while Cu/ZnSODs of B. distachyon had nine β-sheets (Fig. 3d, e, and f) and disulfide bridges (Cys107/Cys196 in XP_003574947 (Fig. 3d); Cys56/Cys145 in XP_003562484 (Fig. 3e), and Cys66/Cys155 in XP_003558478 (Fig. 3f), showing the stability of protein folds as reported in rice [36]. Cu/ZnSODs of eukaryotic organisms consist of highly conserved structures and are composed of two identical subunits. Each subunit includes a β-barrel with eight antiparallel β-strands [42]. The Cu and Zn sites are located at the outside of the β-barrel in the active site channel [35]. In SODs of B. distachyon, β-barrel structures were observed and metal binding sites were located at the outside of the β-barrel structures (Fig. 3d, e, and f) as well, and these are in agreement with the previous data. In Mn/FeSODs, the polypeptide chain contains Nterminal helices and a C-terminal α/β domain. The active sites of Mn/FeSODs are positioned between the N- and C-terminal domains [35, 43]. These data support our findings indicating Mn/FeSODs of B. distachyon had the helix bundles and similar domain structures (Fig. 3a, b and c). The well-defined signature sequences are used for establishing evolutionary relationships among species [44]. Although their locations were different (Fig. 4a and b), conserved signatures (highly conserved regions) were observed in both cytocolic and chloroplastic Cu/ ZnSODs based on the domain analysis of Cu/ZnSODs in B. distachyon. Some residues, including Gly91, Leu92, His93, Gly94, Phe95, His96, Leu97, His98, and Glu99 were observed in chloroplastic Cu/ZnSODs (Fig. 4a), while some residues, including Gly40, Leu41, His42, Gly43, Phe44, His45, Val46, His47, and Ala48 were seen in cytocolic Cu/ ZnSODs (Fig. 4b) of conserved pattern of copper/zinc SOD domains. These residues contribute to β-sheet formation of Cu/ZnSODs in B. distachyon. It can be said that 3D structures of
Fig. 3 Prediction of 3D structures and binding sites of SODs in B. distachyon done by using the 3D LigandSite server (a XP_003568674, MnSOD mitochondrial; b XP_003561161, FeSOD chloroplast; c XP_003557205, FeSOD chloroplast; d XP_003574947, Cu/ZnSOD chloroplast; e XP_003562484, Cu/ZnSOD cytosol; f XP_003558478, Cu/ZnSOD cytosol)
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Fig. 4 Solid ribbon representations of chloroplastic Cu/ZnSOD (XP_003574947) (a) and cytosolic Cu/ZnSOD (XP_003562484) (b) for Pfam patterns of copper/zinc superoxide dismutase domain and mitochondrial MnSOD (XP_003568674) (c) and chloroplastic FeSOD (XP_003561161) (d) for Pfam patterns of iron/manganese superoxide dismutase domain. The structural divergences are indicated as blue empty circles
Cu/ZnSODs in B. distachyon showed structural divergence in domain patterns (Fig. 4a and b). Thus, these divergences could be related with the differences in gene structures of chloroplastic and cytocolic SODs of B. distachyon [12]. Some identified residues such as His195, Ala196, Tyr197, Tyr198, Leu199, Gln200 and Tyr201 in MnSOD of B. distachyon as shown in Fig. 3c and His302, Ala303, Tyr304, Tyr305, Leu306, Asp307 and Tyr308 in FeSOD of B. distachyon as seen in Fig. 3d contribute for the construction of helix structures in B. distachyon. Furthermore, additional β-sheet structure was available in MnSOD of B. distachyon (Fig. 4c) posing effects on the enzyme–substrate interactions and the activity of enzymes in stress conditions. Although, the most of these residues were in same sequences for Cu/ZnSODs (Gly, Leu, His, Gly, Phe, His, X, His, and Y) and Fe/MnSODs (His, Ala, Tyr,
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Fig. 5 Ramachandran plot analyzes of SODs in B. distachyon by using Rampage server (a XP_003568674, MnSOD; b XP_003561161, FeSOD; c XP_003557205, FeSOD; d XP_003574947, Cu/ZnSOD; e XP_003562484, Cu/ZnSOD; f XP_003558478, Cu/ZnSOD)
Tyr, Leu, Z and Tyr), substitutions were occurred in some residues of Cu/ZnSODs (Leu/Val and Glu/Ala) and Fe/MnSODs (Gln/Asp) in B. distachyon. These findings may be related with some genome dynamics, including insertion, deletion, and substitutions in the SOD genes of in B. distachyon. Model Validation For model validation, the Ramachandran plot analyses were performed by utilization of the RAMPAGE server. According to the results, 92.5 %, 93.4 %, 94.1 %, 85.5 %, 94.7 %, and 87.7 % were in favored region, 4 %, 5.1 %, 3.9 %, 9.2 %, 5.3 % and 7.1 % in allowed region and 3.5 %, 1.5 %, 2 %, 5.3 %, 0 % and 5.2 %% in outlier region in MnSOD (XP_003568674), FeSOD (XP_003561161), FeSOD (XP_003557205), Cu/ZnSOD (XP_003574947), Cu/ ZnSOD (XP_003562484), and Cu/ZnSOD (XP_003558478) of B. distachyon, respectively (Fig. 5), suggesting that the 3D models were fairly in good quality. Phylogenetic Analysis In order to understand the phylogenetic relationships of B. distachyon SODs; SOD protein sequences of some grass species (rice, maize, wheat, and Brachypodium) and Arabidopsis
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Fig. 6 Phylogenetic tree of SOD protein sequences in higher plant species, including B. distachyon, A. thaliana, T. aestivum, Z. mays, and O. sativa. Sequence alignment was performed by using ClustalX and phylogenetic tree drawn by Neighbor-Joining method with MEGA 5.1. SODs of B. distachyon are shown in red boxes
were used. The phylogenetic analyzes revealed that plant SODs were separated into two main groups: Mn/FeSOD and Cu/ZnSOD (Fig. 6). As in Brachypodium, Mn/FeSODs and Cu/ ZnSODs were found to be separately clustered in soybean [37] and SODs of rice [36, 45]. Plant MnSODs exhibiting 70 % homology are more distantly related to plant FeSODs indicating separate evolutionary origins [12]. Our results could be related with the separate evolutionary origins of Mn/FeSODs. Mn/FeSOD group was separated into two subgroups: FeSODs and MnSODs. Also, Cu/ZnSOD group was divided into two subgroups: chloroplastic and cytosolic Cu/ZnSODs. In our phylogenetic tree, Mn/FeSODs were grouped together, consisting of both monocots and dicots. Interestingly, MnSODs (dicot) of Arabidopsis were clustered in MnSODs of monocots with the highest bootstrap value (100 %). However, FeSODs of Arabidopsis and Brachypodium were grouped together with 77 % of bootstrap value. It can be proposed that the genes of MnSODs are well conserved and may have been evolved from a common ancestral gene in monocots and dicots. In Cu/ZnSODs, chloroplast Cu/ZnSODs were grouped together with a high bootstrap value (98 %) whereas the cytosolic Cu/ZnSODs were grouped together with a low bootstrap value (64 %). The chloroplastic Cu/ZnSOD (XP_003574947) of B. distachyon and Triticum Cu/ ZnSOD were grouped together with 82 % of bootstrap value. Plant Cu/ZnSODs observed in cytosols, chloroplasts and also peroxisomes; contain a highly homologous group. However, cytosolic and chloroplastic Cu/ZnSODs have the distinctive features with the numbers and positions of introns [12]. This information supports our findings showing the separation of chloroplastic Cu/ZnSODs and cytosolic Cu/ZnSODs. This separation may be affected from gene structure, consisting various numbers and positions of introns (Fig. 2). In conclusion, the molecular structures of SODs are important for understanding of response to oxidative stress in plants. Thus, sequence analyzes and structural models of Mn, Fe and Cu/ZnSODs in B. distachyon were evaluated by employing bioinformatics tools. These analyzes provide insights for molecular functions of SOD isoenzymes related with metal bindings in different cellular organelles. Secondary structural analyzes and predicted binding
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sites of Cu/ZnSODs and Mn/FeSODs of B. distachyon were well conserved, whereas some domain signatures were structurally showed divergences. These binding sites could be important for analyzes of functional site residues in SODs during catalytic processes. Phylogenetic analyzes showed that Fe/MnSODs and Cu/ZnSODs in B. distachyon were distributed separately emphasizing originations of Fe/MnSODs and Cu/ZnSODs from different ancestors. Also, results of this study may contribute to in silico comparative studies about SODs in plants, particularly in grasses.
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