bioinformatic approach: cu, zn superoxide dismutase of ... · of cu, zn sods is demonstrated using...
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INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 4, 2010
© 2010 Ashokan.K.V et al., licensee IPA- Open access - Distributed under Creative Commons Attribution License 2.0
Research article ISSN 0976 – 4402
462
Bioinformatic approach: Cu, Zn superoxide dismutase of Dinoccus
radiodurans
Ashokan. K. V1, Koshti.V.V
2
1- Department of biological science, P.V.P.Colleg, Kavathe Mahankal,
Sangli, Mharashtrea, India
2- Department of Statistics, P.V.P.Colleg, Kavathe Mahankal,
Sangli, Mharashtrea, India
[email protected] doi:10.6088/ijes.00104020005
ABSTRACT Bioinformatic Analysis of Cu, Zn superoxide dismutase protein of Dinococcus radiodurans
strain R1 was performed. The protein’s net positive charge is due to arginine and lysine and
is alkaline with Ip >7 (9.8) and hydrophobicity is 53.5%. Proscan server identified five
functional sites on it like Myristylation site, three phosphorylation sites and an N-
glycosylation site. The secondary structure showed β- turns predominant along with
disulphide bonds and is confirmed by SOPMA. A further study showed that in contain three
domains as Cu, Zn SOD binding, six bladed propeller, TOIB like and SMP-
30/Gluconolaconase/LRE like. The sequence of the domains was analyzed for various
parameters like extinction coefficient, half life, instability index, aliphatic index and grand
average hydropathy (GRAVY).The sequence was then used to generate tertiary structure
which suggest that the Cu, Zn SOD binding site belongs to oxidoreductase fold and metal
binding family, the TOIB like site designated as peptidoglycan associated lipoprotein and
SMP-30 domain assigned as hydrolyzing enzyme. The 3D- structures were evaluated by
Rampage, ProQ and Combinatorial Extension (CE) and visualized by Rasmol.
Keywords: Cu, Zn superoxide dismutase, Dinococcus rdaiodurans, Radiation resistance,
TOIB protein and SMP-30 protein and 3Dstructure
1. Introduction
Radiobiologists are now worked to understanding why Dinococcus radiodurans are
extremely resistant to ionization radiation (IR) (Day and Minton, 1996), by focusing on DNA
repair system expressed during recovery from higher dose of IR (Day and Minton, 1995). D.
radiodurans are also susceptible to damage from prolonged desiccation, while wild type
strain is resistant to both (Mattimore and Battista, 1998). Pearson (2004) has suggested that
the bacterium uses manganese as an antioxidant to protect itself against radiation damage.
High intracellular levels of manganese (II) in D. radiodurans protect protein from being
oxidized by radiation, and proposed protein rather than DNA is the principal target of the
biological action of (ionizing radiation) in sensitive bacteria (Day and Minton, 1995). Studies
in E. coli suggesting that production of oxygen radicals is involved in the mechanism of
bacterial killing in epithelial cells (Battistoni et al. 2000). In bacteria Cu, Zn Superoxide
dismutase are located in the periplasm or anchored to outer membrane (Steinman, 1987,
D’orazio et al.2001, Batistoni, 2000). As superoxide dismutase anion is unable to cross
membranes, Cu, Zn SOD dose not protect bacteria from superoxide generated intracelluarly
(Steinman et al. 1993) but from exogenous source of superoxide. The metal binding property
of Cu, Zn SODs is demonstrated using Haemophilus ducrey (Pacello et al.2001). Cu, Zn
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SOD form a subset of Gram-negative bacteria possesses divalent metal binding N-terminal
extension to uptake enzyme prosthetic metals (Battista et al.2001). Studies by Ken et al.
(2003) showed Cu, Zn SOD is stable at 70º C, resistant to P
H from 2.3 to 12 and sodium
dodocyl sulphate (SDS) under 4%. Danciger at al. (1986) showed that Cu, Zn SOD gene
family, molecular structure and characterization of four Cu, Zn SOD-related pseudo genes
aroused 25 million years ago. The peroxysomal Cu, Zn SOD was characterized from
watermelon showed 70% of homology with cytosolic Cu, Zn SODs. In eukaryotes Cu, Zn
SODs conform to a single structural model that appears to have been strictly preserved
through out the evolution (Battistoni et al.. 1996, Bordo et al. 1999), but analysis of amino
acid sequence of Cu, Zn SODs from different species of Dinococcus showed that this enzyme
exists in multiple form and can be used in identifying Dinicoccus specie (Young and
Young.2001).
From the available literature it has been inferred that there is not much information on
characterization of Cu, Zn SODs protein sequence in D.radiodurans that is involved in
exogenous superoxide protection. Hence the focus of present work is to characterize Cu, Zn
SOD protein (NP_285525.1) of D.radiodurans by using computational tools and servers.
2. Materials and methods
2.1 Sequence analysis of Cu, Zn SOD protein
Diplococcus rdaiodurans strain R1 was selected as the candidate organism for the present
study whose complete genome sequence (gi1798149׀) is available at (www.ncbi.nlm.gov).
The protein sequence of Cu, Zn SOD (NP_285525.1) was downloaded from NCB
(www.ncbi.nlm.nib.gov). Cu, Zn SOD protein sequence was searched for similarity search
using BLAST-P against PDB (Version- 2.2.18+) at Expasy
(http://us.expasy.org/tools/#similarity or http://blast.ncbi.nlm.nih.gov/Blast.cgi). Since the
BLAST algorithm detects both local as well as global alignments, regions of similarity
embedded in otherwise unrelated proteins can be detected (Altschul, 1990). The derived
homologous sequences of Cu, Zn SOD protein were aligned using ClustalW
(http://www.ebi.ac.uk/tools/clustalw/).The Clustal alignment file of the selected sequences
was used for the basic parameters for further creating the phylogenetic tree with Cu, Zn SOD
protein sequence as query sequence. The amino acid composition of Cu, Zn SOD sequence
was computed using CLC sequence viewer version-5 (http://www.clcbio.com).
2.2 Functional Characterization of Cu, Zn SOD
Functional characterization of Cu, ZN SOD sequence was done by submitting the amino acid
sequence of Cu, Zn SOD to prosite (au.expasy.org/proite) and interproscan
(www.ebi.ac.uk/interproscan....).Interproscan is a searchable database providing information
on sequence function as well as annotation and further these sequences are grouped based on
protein signatures (Apweilleret et al.2001). Prosite is a database of protein families and
domains (Flaquet et al.2002). The out put of prosite and interproscan was recorded in terms
of the length of amino acid residues of Cu, Zn SOD protein with specific functional domain.
Further, the results that were obtained from both prosite as well as interproscan were
compared for better interpretation. The obtained domains were analyzed for physicochemical
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parameters, theoretical isoelectric point (Ip), molecular weight, total number of positive and
negative residues, extinction coefficient (EC) ( Gill and Hippel, 1989), half life (Bachmair et
al, 1986), instability index (II) (Guruprasad, 1990), aliphatic index (Ikai, 1980) and grand
average hydropathy (GRAVY) (Kyte and Doolittle,1982) by using Expasy’s PrortParam
prediction server (http://us.expasy.org/tools/protparam.html).The tools SOPM,SOPMA
(Combet et al, 2000) were used for the secondary structure prediction of the domains.
2.3 Secondary structure and 3D structure of Cu, Zn SOD complex
The secondary structure of Cu, Zn SOD protein was obtained from Jpred secondary structure
prediction tool (www.compbio.dundee.ac.uk~www.jp.) by submitting the sequence which
predicts secondary structure using neural network called Jnet (Cuff and Barton, 2000) The
secondary structure prediction is the definition of each residue into either confidence of helix,
confidence of strand (include β-sheet) or confidence of coil secondary structures. The
functional domain predicted by interproscan was submitted to HHpred server
(http://toolkt.tuebingen.mpg.de/hhpred) (Soding, 2005). The generated 3D structure of the
identified domains was visualized by RASMOL (http://www.umass.edu/microbio/rasmol/).
The three dimensional structure of the identified domains were evaluated using the online
server Rampage (Lovell et al l, 2002), ProQ (Crisobal et al, 2001) and CE (Combinatorial
Extension) (Ilya and Philip, 2001).
3. Results
The primary analysis of the protein sequence of the Cu, Zn SOD suggests that the protein
have net positive charge(46) due to the presence of arginine and lysine and negative charge
40 due to Aspartin acid and Glutamine with chemical formula C2137H3428N608O144 S11, having
molecular weight 48295.0 and alkaline with Ip >7 (9.8). The amino acid composition (Table
1) showed hydrophobicity 53.6% and polar amino acid 40.1%. The computed Ip will be
useful for developing buffer system for purification by isoelectric focusing method.
The Cu, Zn SOD protein sequence was searched against PDB database BLAST-P program.
The best 17 sequences on the basis of e-value ranging 4e-21 to 0.005 and score (Bits) ranging
98.6- 38.5, among the 100 sequences retrieved by BLAST-P program against PDB database
was selected (Table 2).The result suggests that protein The primary analysis of the protein
sequence of the Cu, Zn SOD suggests that the protein have net positive charge(46) due to the
presence of arginine and lysine and negative charge 40 due to Aspartin acid and Glutamine
with chemical formula C2137H3428N608O144 S11, having molecular weight 48295.0 and alkaline
with Ip >7 (9.8). The amino acid composition (Table 1) showed hydrophobicity 53.6% and
polar amino acid 40.1%. The computed Ip will be useful for developing buffer system for
purification by isoelectric focusing method.
The Cu, Zn SOD protein sequence was searched against PDB database BLAST-P program.
The best 17 sequences on the basis of e-value ranging 4e-21 to 0.005 and score (Bits) ranging
98.6- 38.5, among the 100 sequences retrieved by BLAST-P program against PDB database
was selected (Table 2).The result suggests that protein sequence with PDB ID 2AQM_A
which belongs to C,Zn SOD an oxidative reductase of Brucella abortus was having highest
degree of similarity to Cu,Zn SOD protein as query sequence indicating through e-value and
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Score (Bits). The phylogram analysis of the selected 17 sequence through ClustalW suggests
that Cu, Zn SOD protein of D.radiodurans was very much similar to Bacillus subtilis (Fig 1).
Both D.radiodurans and B.subtilis form a clad. The function of Cu, Zn SOD was analyzed by
submitting the amino acid sequence of the D.radiodurans to prosite and interproscan server.
Prosite analysis suggested the functionality of Cu, Zn SOD protein with domains identified
for characteristic functionality (Table 3). Jpred program that was used to predict secondary
structure in D.radiodurans suggests that Cu, Zn SOD protein was composed of more β-
sheets than helix and strands (Fig 2).
Table 1: Amino acid composition of Cu, Zn SOD protein
Figure 1: Phylogram analysis of query (Cu,Zn SOD protein of D.radioduran by ClustalW
Amino acid Total number
Percentage
Alanine 47 10.2
Arginine 22 4.8
Asparagine 17 3.7
Aspartic acid 31 6.7
Cysteine 02 0.4
Glutamic acid 18 3.0
Glutamine 09 1.9
Glycine 61 13.2
Histidine 09 1.9
Isoleucine 15 3.2
Leucine 47 10.2
Lysine 24 5.2
Methionine 09 1.9
Phenylalanine 12 2.6
Proline 28 6.1
Serine 21 4.5
Threonine 33 7.1
Tryptophan 03 0.6
Tyrosine 12 2.6
Valine 42 9.1
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Based on the presence of domains in Cu, Zn SOD protein of D.radiodurans the amino acid
sequence of Cu, Zn SOD protein was split into 3 sub units (Table 4). The amino acid
sequence of each domain was analyzed for physico chemical parameters (Table 5). The
domain six bladed propeller, TOIB like and SMP-30 domain showed alkaline nature (Ip .7).
The domain Cu, Zn COD binding showed near to neutral (Ip 6.76).ProtParam of Expasy
computes the extinction coefficient for a range of (1615-31400) wave length, 283nm is
favored because protein absorb strongly there while other substance commonly in protein
solution, do not.
Table 2: Selected 17 sequences of BLAST-P of Cu, Zn SOD protein when searched against
PDB database
Subject ID Identity
In %
Mis
mat
ch
Query Subject
E
value
Bit
Score
In %
Start End Start End
gi׀118137288׀pdb2׀APM׀A
(Brucella abortus) 41.5 84 24 144 02 115 4e-21
98.6
gi ׀10835905׀ pdb ׀EQW1׀ A
(Salmonella typhi) 38.6 86 32 152 10 124 6e-18
87.6
gi׀14277948 ׀ pdb1 ׀IBD׀ A
(Photobacterium leiognathi) 37.9 81 38 158 13 126 5e-17
85.1
gi ׀122920310׀ pdb 2׀GBT ׀A
(Homo sapiens) 36.9 79 29 149 04 110 9e-17
94.1
gi ׀ 12084767׀pdb 1׀E90׀ B
(Bos taurus) 36.4 77 36 156 11 118 9e-16
80.9
gi ׀5269831׀ pdb׀ ITO4 ׀A
( Schistosoma mansoni) 35.3 78 36 154 12 119 3e-13
72.4
gi׀4558010 ׀ pdb 2׀APS׀ A
(Actinobacillus.pleuropneumonia
)
33.8 86 41 161 26 140 4e-13 72.0
gi׀11666687 ׀ pdb 1׀ZQP׀ A
(Haemophilus.ducrey) 35.8 86 41 161 19 133 7e-13
71.2
gi׀ 1065161 ׀pdb 1׀XSO׀ A
(Xenopus levis) 35.5 77 36 155 09 115 9e-13
70.9
gi׀ 118137294 ׀pdb2 ׀AQQ ׀A
(Neisseria meningitidis) 32.4 88 41 161 28 142 1e-12
70.5
gi׀197305046 ׀ pdb3 ׀CE1׀ A
(Cryptococcus liquefaciens) 33.1 88 24 141 02 108 1e-10
63.9
gi׀167013174 ׀ pdb 2׀E46׀ A
(Bombyx mori) 32.5 88 25 144 06 111 5e-10
61.6
gi׀ 66360217 ׀pdb1 ׀U3N ׀A
(Bacillus subtilis) 34.8 64 52 161 33 136 7e-10
61.2
gi׀15826571 ׀ pdb 1׀JK9׀ A
(Sacharmyces cerviseae) 32.5 89 27 144 01 107 9e-10
60.8
gi׀5124721 ׀ pdb 1׀P2S׀ A
(Mycobacterium tuberculosis) 28.0 96 28 145 39 151 8e-7
50.8
gi׀24987493 ׀ pdb 1׀LOQ ׀A
(Methanococcus mazei) 25.5 143 198 372 33 194 1e-04
44.3
gi׀122919993 ׀ pdb2 ׀DSO׀ A
(Staphylococcus aurius) 26.0 125 200 370 48 164 0.001
40.0
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The amino acid sequence of each domain of Cu, Zn SOD obtained from interproscan was
submitted to HHpred server fro 3D structure prediction against PDB templates (Table 6)
individually. In addition to predicting the structure of protein the HHpred was also able to
classify the proteins based of SCOP database. The result of three dimensional structures for
superoxide dismutase, Cu, Zn binding domain
Table3: Functional Characterization of C, Zn SOD protein of D.radiodurans strainR1
by prosite
Sequence Sequence
Length
Function
MYRISTYL N-
myristylation site
14-417aa
An appreciatable number of eukaryotic proteins are acylated by the
covalent addition of myristate Ac C14- saturated fatty acid to their N-
terminal residue via an amid linkage. The sequence specificity of the
enzyme responsible for their modification, myristoyl CoA-protein N-
myristoyl transferase (MMT), has been derived from the sequence of
known N-myristoylated proteins and form studies using synthetic
peptides.
Protein kinase
phosphorylation
site
43-307 aa
In vivo, protein kinase C exhibits preference for the phosphorylation of
serine or threonine residues found close to a C-terminal basic residue. The
presence of additional basic residue at the N- or C- terminal of the target
amino acid enhances the Vmax and Km of the phosphorylation reactions.
N-glycosylation
site
221aa
Potential N-glycosylation sites are specific to the consensus sequence As-
Xaa-Ser/Thr. But the presence of the consensus tripeptide is not sufficient
to conclude that an aspargeine residue is glycosylated to the fact that
folding of the protein plays an important role in the regulation of N-
glycosylation. Presence of proline between Asn and Ser/thr will inhibit N-
glycosylation; similarly 50% of the sites that have a proline C-terminal to
Ser/Thr are not glycosylated.
Casein kinase II
phosphorylation
site
186-189
Casein kinase II (CK-2) is a protein Ser/Thr kinase whose activity is
independent of cyclic nucleotides and calcium Ck-2 phosphorylates many
different proteins. The substrate specificity of this enzyme are
1. Under favorable condition Ser is favored over Thr.
2. An acidic residue (Asn or Glu) must be present 3 residues from
the C-terminal of the phosphate acceptor site.
3. Additional acidic residue in position +1, +2, +4 and +5 increases
phosphorylation rate.
4. Asp is preferred to Glu as the provider of acidic determinants.
Tyrosine kinase
phosphorylation
site
232-
240aa
Substrtae of tyrosine protein are generally characterized by a lysine or
arginine seven residues to the N-terminal side of the phosphorylated
tyrosine residue (Asp or Glu) is often found at either three or four residues
to the N-terminal side of the tyrosine. There are exceptions to this rule
such as tyrosine phosphorylation site of enolase and lipocortin II
of Cu, Zn SOD of D.radiodurans that was predicted by HHpred (Fig 3A) suggests that this
domain contain one chain including one sequence unique to it and having helix-7%, β-sheet
43% and strands-8% and belongs to oxidoreductase Cu,Zn SOD. The domain second consists
of two chains including one unique to it (Fig 3B). The chain unique to it (Chain-A) helix-3%,
β-sheet 51% and strands-9% and belongs to protein Leptospiral antigen Lp49.The domain
third also contain two chains including one unique (chain-A)(Fig 3C). The unique chain
contain helix-4%, β-sheet 51% and strand 9% and belongs to the protein Leptospiral antigen
Lp49. The 3D structure was evaluated by Rampage, ProQ and Combinatorial Extension
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(Table 7) and the result was compared to the standard values for the criteria for good 3D
structure (Table8).
Struc Helices labelled H1, H2, ... and strands by their sheets A, B, ...
Helix Strand
Motifs beta turn gamma turn beta hairpin Disulphides disulphide bond
CSA annotation catalytic residue Residue contacts to ligand to metal
PDB SITE records CUA ZNA PROSITE patterns Low High conservation
Figure 2: Secondary structure prediction of Cu, Zn SOD protein of
D.radiodurans
Table 4: Functional domains Cu, Zn SOD protein of D.radiodurans strain
R1 identified by Interproscan.
Domains Function of domain Position in the
Cu, Zn SOD protein
1 Superoxide Dismutase, Cu,Zn binding 56-200
2 Six bladed β- propeller, TOIB- like 193-446
3 SMP-30/ Gluconolaconase/LRE like protein 191-442
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Table 5: Parameters computed using Expasy’s ProParam tool Protein sequence of Cu,
Zn SOD domains
Amino acid
sequence
Sequenc
e length
M.Wt
Ip
-R
+R
EC
II
AI
GRAVY
Cu, Zn
SOD
C2137H3428N
608O644S11
462
48295.
0
9.08
40
46
34505
21.39
88.87
-0.105
Cu,Zn
SOD
binding
domain
C679H1081N2
11O217S7
155
15876.
7
6.76
14
13
1615
27.94
68.52
-0.257
Six bladed
β-
propeller,
TOIB like
domain
C1208H1942N
32O359S2
254
26868.
8
8.97
23
26
31400
16.11
102.13
-0.046
SMP-
30/Gluco
nolacona
se/LRE
like
domain
C1195H191
9N323O355
S2
252
26555.
4
8.93
22
25
31400
17.22
101.79
-0.026
M.wt: Molecular weight; Ip: Isoelectric point; -R: Number of negative residues; +R Number of positive
residues; EC: Extinction coefficient; II: Instability index; AI: Aliphatic index;
GRAVY: Grand average Hydropathy
Table 6: PDB template (First two hits with maximum
% of identity) obtained using BLAST-P Search.
Accession number PDB Code
IPR001424 1EJ8
IPR011042 3BWS
IPR013658 2DGI
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Table 7: Validation parameters computed for the built 3D structure
Target Template
PDB ode
Rampage percentage residues in the
region of
CR
MSD
(Aº)
ProQ
Favored Allowed Outlier LG
score
Maxsub
IPR001424 1EJ8 134 (97.1%) 4(2.9%) 0
(0.00%)
0.5 4.398 0.373
IPR011042 3BWS 771(95.2%) 39(4.8%) 0
(0.00%)
0.5 6.537 0.401
IPR013658 2DGI 1862(97%) 35(1.8%) 18(0.9%) 0.5 0.506 0.456
Table8: Criteria for a good 3D structure
Rampage
percentage
residus in
favored region
CE RMSD
(Aº)
PoQ
Quality of the
model
LG Score
Maxsub
98
<2
>1.5 >0.1 Fairly good
model
>2.5 >0.5 Very good model
>4 >0.8 Extremely good
model
Figure 3A: Cu,Zn SOD binding domain
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Figure 3B: six bladed beeta propellers, TOB
Figure 3C: SMP-30/Gluconolaconase/LRE-like protein domain
4. Discussion
Until relatively recently, Cu, Zn SOD was considered to be an almost exclusively eukaryotic
enzyme, and its presence in bacteria was thought to be an exception rather than a rule. Unlike
eukaryotes Cu, Zn SODs are located in the periplasm or anchored to the outer membrane
(Steiman, 1987, D’orazio et al. 2001). Radiation and oxidative stress resistant bacterium
D.radiodurans possess redundant copies of the Cu, Zn SOD gene sod C (Battista et al, 2001).
In eukaryote Cu, Zn SOD conforms to single structural model, but in prokaryotes Cu, Zn
SOD occurs in great variation from species to specie, hence individual enzyme variant may
exhibit unique properties (Batistoni et al.1996, Bordo et al. 1999). In this context exploring
the structural and functional role of Cu, Zn SOD in D.radiodurans is appropriate.
D.radiodurans is the most radiation resistant organism yet discovered (Day and Minton,
1996). The hypothesis of protein enzyme is responsible for radiation resistance out weighs
the DNA repair hypothesis (Day et al.. 1954).Cloning sequence information with 3D
structure gives invaluable insight for the development of effective rational strategies for
experiments such as site directed mutagenesis, studies of radiation stress resistant mutations,
or the structure based design of specific inhibitors. The studies to aim function-structure
aspects of protein will assists to find out the mechanism of radiation resistance in bacterium
like D.radiodurans in future. The analysis of the Cu, Zn SOD sequence in D.radiodurans
suggests that it contain three domains. One of the domains reveals structure related to the Cu,
Zn SOD domains of Brucelli. The other two domains reveal homology structure to bacterial
antigen Lp49 protein. The BLAST-P search followed by multiple alignments using ClustalW
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reveals that D.radiodurans closely related to Bacillus subtilis form separate clad, indicating
divergence of D.radiodurans and Bacillus subtilis occur at the same period. The divergence
of D.radiodurans and bacillus may occur due to the differential environmental stress along
with natural selection. The sequence length of D.radiodurans is 3 times longer than other
bacteria selected.
The secondary structure prediction of Cu, Zn SOD showed the protein contains more β-
sheets than helix. The functional characterization of Cu, Zn SOD predicted through prosite
showed various components like N-myristylation site, phosphorylation site and N-
Glycosylation site. All these are markers of high activity of enzyme in stress environment.
The 3 dimensional structures evaluated through HHpred and their visualization using
Rasmole reflected the association of the Cu, Zn SOD to cope with stress environment. The
three domains of Cu, Zn SOD i.e. Cu, Zn SOD binding, SMP-30 and TOIB are some way
involved in protecting the free radical attack, particularly from exogenous source in the case
of Dinococcus rdaiodurans.
5. References
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Gouzy, j., Hermjakob, H., Hulo, N., Nonassen, I., Khan, D., Kanapin, A., Karavidopoulou,
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