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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

akvsangli@gmail.com 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.

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