American Journal of Plant Sciences, 2012, 3, 1311-1321
http://dx.doi.org/10.4236/ajps.2012.39158 Published Online September 2012 (http://www.SciRP.org/journal/ajps) 1311
Comparative Analysis of Structure and Sequences of
Oryza sativa Superoxide Dismutase
Aiyar Balasubramanian1, Sonali Das1, Anjana Bora1, Susmita Sarangi1, Asit B. Mandal2*
1SUB-Distributed Informatics Centre, Central Agricultural Research Institute, A & N Islands, India; 2Division of Crop Improvement,
Central Research Institute for Jute and Allied Fibres, Barrackpore, India.
Email: *amandal2@rediffmail.com
Received May 21st, 2012; revised June 8th, 2012; accepted July 23rd, 2012
ABSTRACT
One of the major classes of antioxidant enzymes, which protect the cellular and subcellular components against harmful
reactive oxygen species (ROS), is superoxide dismutase (SOD). SODs play pivotal role in scavenging highly reactive
free oxygen r adicals and protecting cells from toxic effects. In Oryza sativa three types of SODs are available based on
their metal content viz. Cu-Zn SOD, Mn SOD and Fe SOD. In the present study attempts were made to critically assess
the structure and phylogenetic relationship among Oryza sativa SODs. The sequence similarity search using local
BLAST shows that Mn SODs and Fe SODs have greater degree of similarity compared with that of Cu-Zn SODs. Th e
multiple alignment reveals that seven amino acids were found to be totally conserved. The secondary structure shows
that Mn SODs and Fe SODs have similar helixes, sheets, turns and coils compared with that of Cu-Zn SODs. The com-
parative analysis also displayed greater resemblance in primary, secondary and tertiary structures of Fe SODs and Mn
SODs. Comparison between the structure and sequence analysis reveals that Mn SOD and Fe SOD are found to be
closely related whereas Cu-Zn SOD evolves independently.
Keywords: Oryza sativa; Primary Structure; Sequence Analysis; Tertiary Structure; S uperoxid e Dismu tase
1. Introduction
Protein sequence comparison is the most powerful tool in
characterizing protein sequences because of the enor-
mous amount of information kept in the protein domain
throughout the evolutionary process. For many protein
sequences, even evolutionary history can be traced back
to 1 - 2 billion years ago. Sequence comparison is most
effective in homologous protein, which always shares the
common active sites or binding domains. Comparative
study of protein structures enables the study of functional
relationships between proteins and it bears immense im-
portance in homology search and threading methods in
structure prediction. Multiple structure alignment of pro-
tein is needed in order to group proteins into families,
which enables a subsequent analysis of evolutionary is-
sues. SODs (Superoxide dismutase) constitute the first
line of defence against reactive oxygen species (ROS).
SODs belong to a large and ubiquitous family of metal-
loenzymes in aerobic organisms [1]. The scavenging ca-
pacity of superoxide radicals (2
O) is achieved through
an upstream enzyme SOD, which catalyses the dismuta-
tion of superoxide to hydrogen peroxide (H2O2). SODs
are omnipresent in all aerobic organisms and in all sub-
cellular compartments susceptible to oxidative stress [2].
SODs are classified based on the metal cofactor embo-
died in the active site of the enzyme. A new type of SOD
with “Ni” in the active centre (Ni SOD) has been de-
scribed in Streptomyces [3] recently.
The majority of the research ha s focused on eith er me-
dicinal or agricultural applications; however, the origin
of rice (Oryza sativa) has become an important model
system with immediate practical applications because of
its economic and nutritional importance worldwid e [4,5].
Rice has several advantages as a model plant. It has a
relatively small genome (~430 Mb) that has been almost
completely sequenced [6]. Abiotic stress is the major en-
vironmental constraint to rice production in non-irrigated
rice areas [7]. SODs are metalloenzymes in aerobic or-
ganisms that play a crucial role in protecting organisms
against ROS in rice [8]).
The present investigation is an attempt to analyze the
sequence of Oryza sativa SODs by using computational
tools and techniques in order to understand the biological
functions and evolutionary relationships among the Oryza
sativa SODs. By understanding the sequence, structure
and function relationships between SODs in future new
proteins having all possible characters of SODs can be
designed to produce cultivars tolerant to reactive oxygen
*Corresponding a uthor.
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Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
1312
species (ROS).
2. Materials and Methods
In this study bioinformatics tools, software and methods
were used for the sequences comparison of Oryza sativa
SODs. This includes sequence retrieval, local BLAST
database creation and BLAST search, BioEdit, Port-Pa-
ram tool, MEGA phylogenetic tree construction, ANTH-
EPORT-SOPMA, SWISS-MODEL, YASARA, PRO-
CHECK-COMP, Ramachandran plot analysis and PTF
database.
2.1. Sequence Retrieval
In order to analyze protein sequences and structures the
amino acids sequences of the protein of interest is most
essential. Protein sequences can be searched from a vari-
ety of protein primary sequence databases viz. (PIR,
MIPS, SWISS PROT, TrEMBL, NRL-3D [9] (Attwood
and Parry-Smith, 2002)). SWISS-PROT/TrEMBL data-
base used for retrieving the amino acid sequences of
Oryza sativa SODs. SWISS-PROT is an annotated pro-
tein sequence database established in 1986 and main-
tained collaboratively by the Swiss Institute for Bioin-
formatics (SIB) and the European Bioinformatics Insti-
tute (EBI). The data in SWISS-PROT are derived from
translations of DNA sequences from the EMBL nucleo-
tide sequence database, adapted form the Protein Identi-
fication Resource (PIR) collection, extracted from the
literature and directly submitted by researchers. TrEMBL,
a computer-annotated supplement to SWISS-PROT, ac-
companies SWISS-PROT. Amino acids sequences of
Oryza sativa SODs were retrieved from SWISS-PROT/
TrEMBL available at www.expasy.org.
2.2. Sequence/Primary Structure Analysis
The exact nature of the information encoded in primary
structure is still unclear. Detailed fold ing studies have re-
vealed more and more complexities, making it under-
stand that the sequence to structure relation is a very
complicated problem. Using sequence analysis techni-
ques, attempt was made to identify similarities between
novel query sequence and database sequences, whose
structures and functions have been elucidated. The straight
forward at high levels of sequence identity, where rela-
tionships are distinctly clear, but below 50% identity it
becomes increasingly difficult to establish relationships
reliably [8].
2.2.1. Local BLAST
Local BLAST has been created by using BioEdit. Local
BLAST (Basic Local Alignment Search Tool) search is
often used as the most convenient method for detecting
homology of a biological sequence to existing character-
ized sequences. Local BLAST looks for homology by
searching for locally aligned regions of identity and
similarity between a query sequence in a local database.
Local BLAST database was created by using file con-
taining all the sequences of Oryza sativa SODs in
FASTA format. In BioEdit, from the “Accessary applica-
tion” menu “BLAST” was chosen. Then ‘Create local
database’ was selected. The rest of the things were auto-
matic. The database automatically placed into the data-
base folder for the BioEdit install directory. This local
BLAST has been used to find out the sequence similarity
between Oryza sativa SODs. The query sequence of
every SODs were given to local BLAST for BLAST
search against all Oryza sativa SODs that were present in
the local BLAST databases.
2.2.2. Pr otParam
ProtParam is a tool, which allows the computation of
various physical and chemical parameters for a given
protein stored in SWISS-PROT or TrEMBL for a user
entered sequence. The computed parameters include the
molecular weight, theoretical isoelectric point (pI),
amino acid composition, atomic composition, extinction
coefficient, estimated half-life, instability index, alip hatic
index and grand average of hydropathicity (GRAVY).
The protein can be specified as a SWISS-PROT of TrE-
MBL accession number or ID or in the form of raw se-
quence. If the accession number of a SWISS-PROT or
TrEMBL entry is provided, then we prompt with an in-
termediary page that allows selecting the portion of the
sequence on which analysis is to be performed. The
choice includes a selection of mature chains or peptides
and domains from the SWISS-PROT/TrEMBL table, as
well as the possibility to enter start and position in two
boxes. By default the complete sequence will be ana-
lyzed. PortParam tool has been used for analyzing phy-
siochemical properties of Oryza sativa SODs. The amino
acids sequence of Oryza sativa SODs in FASTA format
were given to ProtParam tool available at www.expasy.
org. The physiochemical properties of Oryza sativa
SODs were given.
2.2.3. BioEdit
BioEdit is a biological sequence editor that runs in Win-
dows and is intended to provide basic functions for pro-
tein and nucleic sequence editing, alignment, manipula-
tion and analysis. Hydrophobic amino acids tend to occu r
in the interior of globular proteins, while at the surface of
a protein one will preferen tially find hydroph ilic residues.
The hydrophobicity scale or related scale are frequently
used for the prediction of antigenic epitopes. Mean hy-
drophibicity profiles are generated using the general
method of Kyte-Doolitte. Kyte-Doolitte compiled a set of
“hydropathy scores” for the 20 amino acids based upon
Copyright © 2012 SciRes. AJPS
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase 1313
compilation of experimental data from the literature. A
window of defined size is moved across a sequence, the
hydropathy scores are summed along the window, and
the average is taken for each position in the sequence.
The mean hydrophobicity value is plotted for the middle
residue of the window. Hydrophobic moment profiles
plot the hydrophobic moment of segments of defined
length along the sequence. For example, if the window
size is 21 residues, the plotted value at a residue is the
hydrophobic moment of the window of 10 residues on
either side of the current residue. Hydrophobic moment
is calculated according to Eisenberg method [10],
mH = {[SHnsin(dn)]2 = [SHncos(dn)]}1/2
where “mH” is the hydrophobic moment, “Hn” is the hy-
drophobicity score of residue “H” at position n, d = 100
degrees, “n” is position with in the segment, and each hy-
drophobic moment is summed over a segment of the
same defined window length. BioEdit has been used to
generate the hydrophobicity plot of Oryza sativa SODs.
The file containing Oryza sativa SODs were opened in
BioEdit alignment window. In the sequence analysis
menu, protein sequence, Kyte-Doolitte hydrophobicity
plot were chosen. Subsequently the hydrophobicity plot
for Oryza sativa SODs was obtained.
2.2.4. T-COFFEE
Tree-based Consistency Objective Function for Align-
ment Evaluation (T-COFFEE) is a new progressive
method for sequence alignment. Multiple alignments are
essential pre-requisites for further analyses of protein
families such as homology modeling or phylogenic re-
construction, or simply used to illustrate conserved and
variable sites within a family. Those alignments may be
further used to derive profiles or hidden Markov models
[11] that can be used to scour databases for distantly re-
lated members of the family.
T-COFFEE combine signals from heterogeneous sour-
ces into a unique consensus multiple sequence alignment
[12]. T-COFFEE has two main features. Firstly, it pro-
vides a simple and flexible means of generating multiple
alignments using heterogeneous data sources that are
provided to T-COFFEE via a library of pair-wise align-
ments. Secondly T-COFFEE is the optimization method,
which is used to find the multiple alignments that b est fit
the pair-wise alignments in the input library.
2.2.5. MEGA
Molecular Evolutionary Genetic Analysis (MEGA) cre-
ating a multiple sequence alignments using Clustal W or
Clustal X. The main use of this software is to estimate
evolutionary distances and to build the phylogenetic tree
of multiple protein and nucleotid e sequences. MEGA has
been used for the phylogenetic analysis of Oryza sativa
SODs. The file containing Oryza sativa SODs amino
acid sequences in FASTA were opened in MEGA win-
dow and were converted to MEGA file. Then by multiple
sequence alignment MEGA alignments file was created.
By MEGA alignment file the phylogenetic tree of Oryza
sativa SODs was constructed.
2.3. Secondary Structure Analysis
Antheprot
A graphic programme was developed to calculate the
secondary structure content of proteins from their circu-
lar dichroism spectrum. All information concerning
analysis and results are given on a single screen. The
percentages of secondary structure and statistical pa-
rameters are provided. The secondary structure predic-
tion called “SOPMA” which means Self Optimized Pre-
diction from Multiple Alignment in ANTHEPROT.
SOPMA is an improvement of SOPM method. These
methods are based on the homologue method. The im-
provement takes place in the fact that SOPMA takes into
account information from an alignment of sequences be-
longing to the same family. If there are no homologous
sequences the SOPMA prediction is the SOPM one. The
first step of the SOPM is to build sub-databases of pro-
tein sequences and their known secondary structures
drawn from “DATABASE. DSSP” by 1) making binary
comparisons of all protein sequences and 2) taking into
account the prediction of structural classes of proteins.
The second step is to submit each protein of the sub-
database to a secondary structure prediction using a pre-
dictive algorithm based on sequence similarity. The third
step is to iteratively determine the predictive parameters
that optimize the prediction quality on the whole sub-
database. The last step is to app ly the final parameters to
the query sequence. When a sequence is submitted to
SOPMA from within ANTHEPROT, it will automati-
cally provides to NPS@ web server (http://pbil.ibcp.
fr/NPSA) through the Internet. The SOPMA method is
able to also predict the turn state but accuracies are given
only for four states (Helix, Sheet Coil and Turn). The
Oryza sativa SODs sequences were given to ANTHE-
PROT-SOPMA method for secondary structure predict-
tion and for analysis. The secondary structure of Oryza
sativa SODs were displayed in ANTHEPROT graphic
viewer . The seco ndary stru cture con tent statis tics of Ory-
za sativa SODs were taken by selecting “Details menu”
in the graphic viewer.
2.4. Tertiary Structure Analysis
2.4.1. Swiss-Model
In order to analyze the functional properties of the pro-
tein structure of the protein of our interest was felt essen-
tially required. By submitting th e sequence to the servers
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Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
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one can get the structure of the proteins through email.
SWISS-MODEL used to predict the structure of Oryza
sativa SODs available at
http://www.expasy.org/swissmod/swiss-model.html.
SWISS-MODEL is a server for automated comparative
modeling of the three dimensional (3D) structures.
SWISS-MODEL provides several levels of user interact-
tion through World Wide Web interface: in the “first ap-
proach mode” only an amino acid sequence of a protein
is submitted to build a 3D model. Template selection,
alignment and model building are done completely auto-
mated by the server. In the “alig nment mode” the model-
ing process is based on a user defined target template
alignment. Complex modeling tasks can be handled with
the “project mode” using Deep View (SWISS-pdb Vie-
wer), an integrated sequence to structure workbench. All
the models are sent back via email with detailed model-
ing report.
2.4.2. YASARA
The structure given by the SWISS-MODEL was visual-
ized using visualizing tools and was analyzed. YASARA
used to visualize tertiary structu res of Oryza sativa SODs.
YASARA is a molecular graphics, molecular modeling
and molecular simulation programme. With an intuitive
user interface, photorealistic graphics and support for
affordable shutter glasses, utostereoscopic displays and
input devices. YASARA has been used to visualize terti-
ary structure, superimposition of SODs and to find the
metal ion present in the active site of every Oryza sativa
SODs isoform.
2.4.3. PROCH E CK- COMP
PROCHECK-COMP is meant for comparing the residue
by residue geometry of a set of closely related structures,
such as separate members of a family or models of the
same structure saved during different stages of refine-
ment. It outputs a number of PostScript files showing the
comparisons, including residue by residue Ramachan-
dran plots and comparison of the different secondary
structure elements in each PDB file. This programme is
used for protein structure comparison. It compares resi-
dues by residue geometry of closely related proteins stru-
ctures. It is used to compare closely related structures
such as, membrane of a family of proteins, models of a
structure saved at different stages of refinement and ho-
mology model and structure. This programme is now
available with PROCHECK programme. The PDB file
was uploaded to
http://www.jcsg.org/scripts/prod/validation/sv3.cgi
server available at JCSG database. Then the results were
produced in postscript file, and subsequently seen by
using GS vi ewer.
2.4.4. T h e Ramachand r a n Plot
Ramachandran plot developed by Gopalasamudram Na-
rayana Ramachandran [13] is a way to visualize dihedral
angles φ against ψ of amino acids residues in protein
structure. It shows the possible conformations of φ and ψ
angles for a polypeptide. In a polypeptide the main chain
N-C alpha and C alpha-C bonds relatively are free to
rotate. These rotations are represented by the torsion an-
gles phi and psi, respectively. In the diagram the white
areas correspond to conformations where atoms in the
polypeptide come closer the sum of their van der Waals
radii. The red reg ion s corr espo nd to conformatio n s wh er e
there are no steric clashes, i.e. these are the allowed re-
gions, namely the alpha-helical and beta-sheet conforma-
tions. The yellow areas show the allowed regions if
slightly shorter v an der Waals radii are u sed in the cal cu-
lations, i.e., the atoms are allowed to come a little closer
together. This brings out an additional region, which
corresponds to the left-handed alpha helix. Disallowed
regions generally involve steric hindrance between the
side chain C-beta methylene group and main chain atoms.
Glycine has no side chain and therefore can adopt phi
and psi angles in all four quadrants of the Ramachandran
plot.
2.4.5. Chi m era
Chimera software was used to compare the 3D structure
of macromolecules by superimposing one structure with
another. All structures of Oryza sativa SODs were
opened. Then tools, structure comparison were chosen.
Finally the aligned sequences of SODs structure were
seen in the chime alignment window.
2.4.6. PTF Database
PTF is an automated protein function predicting server,
which was available at http://dragon.bio.purdue. edu/pfp
/pfp.html. The sequence submitted was queried with an
interactive PSI-BLAST against UniProt. These results
were cross-referenced to the Gene ontology annotation
file. Then subsequently results were listed as the top 10
most probable Gene ontology annotations in the biologi-
cal process, molecular function and cellular component
categories. Oryza sativa SODs sequences were given to
PTF server, and the cellular location of SODs and func-
tions were got via email.
3. Results and Discussion
The process of comparison of Oryza sativa SODs (in-
cluding) sequence retrievals, Local BLAST search, phy-
siochemical property analysis, multiple sequence align-
ment, phylogenetic analysis, secondary structure and ter-
tiary structure prediction and analysis, cellular locations
and function prediction) showed substantial sequence
similarities.
Copyright © 2012 SciRes. AJPS
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
Copyright © 2012 SciRes. AJPS
1315
Through local BLAST amongst Oryza sativa SODs,
each comparison is given a score reflecting the degree of
similarity between the query and the Oryza sativa SODs
sequence in local BLAST database (Tabl e 1 ). The higher
the score, the greater the degree of similarity. Each com-
parison also gives expected value and identities.
Table 1. Sequence similarity between Oryza sativa SODs. (a) Local BLAST result for P28756 Cu-Zn SOD; (b) Local BLAST
result for P28757 Cu-Zn SOD; (c) Local BLAST result for Q4TUB3 Cu-Zn SOD; (d) Local BLAST result for Q76MX3
Cu-Zn SOD; (e) Local BLAST result for Q43008 Mn SOD; (f) Local BLAST result for Q43121 Mn SOD; (g) Local BLAST
result for Q43803 Mn SOD; (h) Local BLAST result for Q7GCN0 Mn SOD; (i) Local BLAST result for Q4VQ67 Fe SOD; (j)
Local BLAST result for Q52WX4 Fe SOD; (k) Local BLAST result for Q5VSB7 Fe SOD; (l) Local BLAST result for
Q9ZWM8 Fe SOD.
(a)
Oryza sativa SODs Score Expect Identity (%) Positive (%)
Q4TUB3 Cu-Zn SOD 313 7e–090 100 100
P28757 Cu-Zn SOD 280 1e–079 88 93
Q76MX3 Cu-Zn SOD 211 1e–059 64 77
(b)
Oryza sativa SODs Score Expect Identity (%) Positive (%)
Q4TUB3 Cu-Zn SOD 2 8 0 1e–090 88 93
P28756 Cu-Zn SOD 2 80 1e–079 88 93
Q76MX3 Cu-Zn SOD 207 8e–058 64 148
(c)
Oryza sativa SODs Score Expect Identity (%) Positive (%)
P28756 Cu-Zn SOD 313 7e–090 100 100
P28757 Cu-Zn SOD 280 1e–079 88 93
Q76MX3 Cu-Zn SOD 211 7e–059 64 77
(d)
Oryza sativa SODs Score Expect Identities (%) Positive (%)
Q4TUB3 Cu-Zn SOD 211 1e–058 64 77
P28756 Cu-Zn SOD 211 1e–058 64 77
P28757 Cu-Zn SOD 207 7e–057 64 77
(e)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q7GCNO Mn SOD 469 e–136 99 99
Q43121 Mn SOD 469 e–136 99 99
Q43803 Mn SOD 464 e–135 98 99
Q5VSB7 Fe SOD 464 3e–02 8 37 51
Q9ZWM8 Fe SOD 106 3e–027 37 51
Q52WX4 Fe SOD 90.9 2e–022 41 59
Q4VQ67 Fe SOD 69.3 5e–136 36 46
(f)
Oryza sativa SODs Score Expect Identity (%) Positive (%)
Q7GCNO Mn SOD 472 e–137 100 100
Q43008 Mn SOD 46 9 e–136 99 99
Q43803 Mn SOD 46 7 e–136 98 99
Q5VSB7 Fe SOD 110 3e–028 37 51
Q9ZWM8 Fe SOD 106 3e–027 37 51
Q52WX4 Fe SOD 90.9 2e–022 41 59
Q4VQ67 Fe SOD 69.3 5e–0 16 36 46
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
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(g)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q7GCNO Mn SOD 472 e–136 98 99
Q43121 Mn SOD 467 e–136 98 99
Q43008 Mn SOD 464 e–135 98 99
Q5VSB7 Fe SOD 110 3e–02 8 37 51
Q9ZWM8 Fe SOD 106 3e–027 37 51
Q52WX4 Fe SOD 90.9 2e–022 41 59
Q4VQ67 Fe SOD 69.3 5e–016 36 46
(h)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q43121 Mn SOD 472 e–137 100 100
Q43008 Mn SOD 469 e–136 99 99
Q43803 Mn SOD 467 e–136 98 99
Q5VSB7 Fe SOD 110 3e–02 8 37 51
Q9ZWM8 Fe SOD 106 3e–027 37 51
Q52WX4 Fe SOD 90.9 2e–022 41 59
Q4VQ67 Fe SOD 69.3 5e–016 36 46
(i)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q5VSB7 Fe SOD 359 e–103 98 99
Q9ZWM8 Fe SOD 354 e–102 98 98
Q52WX4 Fe SOD 128 6e–034 98 98
Q7GCN0 Mn SOD 69.3 5e–016 36 46
Q43803 Mn SOD 69.3 5e–016 36 46
Q43121 Mn SOD 69.3 5e–016 36 46
Q43008 Mn SOD 69.3 5e–016 36 46
(j)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q5VSB7 Fe SOD 295 2e–08 4 100 100
Q9ZWM8 Fe SOD 288 3e–082 98 98
Q4VQ67 Fe SOD 128 4e–03 4 98 98
Q7GCN0 Mn SOD 90.9 9e–023 41 59
Q43803 Mn SOD 90.9 9e–0123 41 59
Q43121 Mn SOD 90.9 9e–0123 41 59
Q43008 Mn SOD 90.9 9e–0123 41 59
(k)
Oryza sativa SODs Score E value Identity (%) Positive (%)
Q9ZWM8 Fe SOD 520 e–152 99 99
Q4VQ67 Fe SOD 359 e–103 98 99
Q52WX4 Fe SOD 295 4e–084 100 100
Q7GCN0 Mn SOD 110 3e–028 37 51
Q43803 Mn SOD 110 3e–028 37 51
Q43121 Mn SOD 110 3e–028 37 51
Q43008 Mn SOD 110 3e–028 37 51
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Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
Copyright © 2012 SciRes. AJPS
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(l)
Oryza sativa SODs Score E value Identity (%) Positive (%)
57VSB7 Fe SOD 520 e–152 99 99
Q4VQ67 Fe SOD 354 e–102 98 98
Q52WX4 Fe SOD 288 6e–082 98 98
Q7GCN0 Mn SOD 106 3e–027 37 51
Q43803 Mn SOD 106 3e–027 37 51
Q43121 Mn SOD 106 3e–027 37 51
Q43008 Mn SOD 106 3e–027 37 51
From the investigation results of local BLAST search
of Oryza sativa Mn SODs have an average of 63.5%
similarity to one another. This is in conformity with the
findings of Bowler et al., [2]. Accord ing to Bowler plant
Mn SODs have been reported to have about 65% se-
quence similarity to each other. With the detailed study,
the distribution of SODs both at the sub-cellular level
and at the phylogene tic level it was observed that only in
plants all three different types of SODs co-exist. Com-
parison to deduce amino acid sequences from the three
types of SODs suggests that Mn SODs and Fe SODs are
more efficient. SODs and these enzymes most probably
have arisen from the same ancestral enzymes, whereas
Cu-Zn SODs have evolved separately in eukaryotes. This
view has also been found to be corroborative to the find-
ings of Smith and Doolitte.
Multiple sequence alignment of Oryza sativa SODs
reveals that seven amino acids (N-150, G-165, I-177,
G-200, G-202, L-206 and L-236) were totally conserve,
whereas the multiple sequence alignments between Mn
SODs and Fe SODs showed greater number of conserved
amino acids residues. Th is is in conformity with the find-
ing of Sch and Kardinahl [14], 2003. Multiple sequence
alignments could not differentiate decisively between Mn
SODs and Fe SODs, especially as the pattern and type of
metal binding residue s are absolutely identical.
The phylogenetic tree (Figure 1) shows that Cu-Zn
SODs evolved independently in Oryza sativa SODs.
Short distance between branch points of the Mn SODs
and Fe SODs suggests a common phylogenetic origin
and likely frequent horizontal gene transfer during early
evolution. Within the major SODs domain, the Cu-Zn
SODs are descendants from a common root. This is in
conformity with the findings of Sch and Kardinahl [14]
in Arabidopsis thaliana genome analysis. The phyloge-
netic tree pattern is supported by the hypothesis proposed
by Martin and Fridovich, 1981. Th e hypo thesis is th at th e
Fe SODs are of great antiquity and that the Cu-Zn SODs
evolved independently of Mn SODs and Fe SODs.
Cu-Zn SODs originated in the eukaryotes and the eu-
karyotes gene was transferred into the prokaryotes [15].
The results show that the Cu-Zn SODs and Fe SODs
have similar physiochemical properties like amino acid
contents, theoretical isoelectric point, number of posi-
tively charged residues and number of sulphur atoms etc.
The physiochemical properties of the Photobacter leiog-
nuthi Cu-Zn SOD were very similar to those of the com-
parable to CU-Zn SOD, Fe SOD enzymes from other
sources, especially those of teleost fish [15].
Doolitte-Kyte hydrophobicity plot of Oryza sativa
SODs (Figure 2) displayed its hydrophobic character,
which may be useful in predicting membrane-spanning
domains, potential antigenic sites and regions that are
likely exposed on the protein’s surface. The Mn SODs
and Fe SODs have a greater number of hydrophilic resi-
dues than Cu-Zn SODs.
Tertiary structure of Oryza sativa Mn SODs and Fe
SODs had higher similarities. Both SODs contain an al-
pha/beta fold, which differs from the greek key bête-
barrel of Cu-Zn SODs. This might be due to the greater
sequence similarities between Mn SODs and Fe SODs.
Tertiary structure of protein depends upon the primary
structure of a protein. So Mn SODs shows greater simi-
larity. This is in conformity with the finding of Stallings
et al., [16], which showed high similarity among three
tertiary structures of Mn SODs and Fe SODs.
YASARA shows that Mn SODs and Fe SODs are typi-
cally observed to be homodimers and homotetramers.
Each 200-residues monomer is bound to be metal ion.
The active sites of SODs are specific for their respective
metal ions and for the superoxide anion. Those exhibit a
conserved structure that consists of a group of metal-
binding residues enclosed by shell of residues. Although
both enzymes have the ability to bind either Mn or Fe,
the corresponding metal ion principally governs the na-
tive SOD enzyme activity.
From the PTF database it is understood that Oryza sa-
tiva Mn SODs and Fe SODs were present in the mito-
chondria, whereas CU-Zn SODs were present in the
apoplast. The molecular function of Cu-Zn SODs also
differs from MnSODs and FeSODs. This reflects that
specialization of functions among the SODs may be due
to the influence of cellular or tissue localization of the
enzyme. This observation is supported by Alscher et al.,
[17].
The Ramachandran plot of Oryza sativa SODs dis-
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
1318
Figure 1. Phylogenetic tree of Oryza sativa SODs in rectangular shape obtained by using MEGA. The tree shows the calcu-
lated evolutionary relationships of known Oryza sativa SODs. The length of horizontal lines connecting on sequence to an-
other is proportional to the estimated ge ne tic distance between the se quences.
Figure 2. Kyte-Doolitte hydrophobicity plot for Oryza sativa SODs obtained by BioEdit. The plot has amino acid sequence of
Oryza sativa SODs on its X-axis and degree of hydrophobicity on its Y-axis.
played that the Mn SODs (Figure 3(b)) and Fe SODs
(Figure 3(c)) had a greater number of residues in fa-
voured and allowed regions, whereas Cu-Zn SODs (Fig-
ure 3(a)) have a large number of residues in disallowed
residues. The molecular function of Mn SODs and Fe
SODs were found to be similar i.e., superoxide dismutase
activity. Function of every protein is determined by its
tertiary structure.
The superimposing of structure by using Chimera
shows that Mn SOD and Fe SOD have highly similar
Copyright © 2012 SciRes. AJPS
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase 1319
(a)
(b)
Copyright © 2012 SciRes. AJPS
Comparative Analysis of Structure and Sequences of Oryza sativa Superoxide Dismutase
1320
(c)
Figure 3. (a) Ramachandran plot of Oryza sativa Cu-Zn SODs. The Ramachandran plots of Oryza sativa SODs shows the
amino acid residues present in the
most favoured,
generously allowed region,
allowed and
disallowed region
of Ramachandran plot; (b) Ramachandran plot of Oryza sativa Mn SODs. The Ramachandran plots of Oryza sativa SODs
shows the amino acid residues present in the
most favoured,
generously allowed region,
allowed and
disal-
lowed region of Ramachandran plot; (c)-Ramachandran plot of Oryza sativa Fe SODs. The Ramachandran plots of Oryza
sativa SODs shows the amino acid residue s present in the
most favoured,
generously allowed region,
allowed and
disallowed region of Ramachandran plot.
structures, whereas the structure of Cu-Zn SODs was
found to be totally different from other two SODs. The
functions of SODs also depend on the metal atoms pre-
sent in the active site. In Mn SODs and Fe SODs only
single metal is present. Whereas in Cu-Zn SODs “Cu”
and “Zn” atoms were present so the active sites act inde-
pendently. The function of Cu-Zn SODs is also found to
be differing from other two SODs.
The comparative analysis of Oryza sativa SODs shows
great similarity in primary, secondary and tertiary struc-
tures of Fe SODs and Mn SODs. Comparison of se-
quence and structure of Oryza sativa SODs reveals that
Mn SOD and Fe SOD groups are closely related,
whereas the Cu-Zn SOD enzymes apparently has evolved
independently. Mn SODs and Fe SODs have similar ac-
tive site. By understanding the sequence, structure and
function relationship between SODs in future someone
can design new proteins having all possible characters of
SODs to produce rice cultivars tolerant to reactive oxy-
gen intermediates species.
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