1 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

ISSN: 2277-3827

Original article

Homology modeling of a unique extracelluar glutaminase free L-asparaginase

from novel marine Actinomycetes 1Senthil Kumar.M*,

2Selvam.K and

3Singaravel.R

1. Dept of Biotechnology, Bharathiar University, Coimbatore, India.

2. Dept of Biotechnology, Dr.NGP college of Arts and Science, Coimbatore, India.

3. Central Institute of Brackishwater Aquaculture (CIBA), Chennai, India.

*Corresponding Email: pearlsen78@gmail.com

Received 15 March 2012; accepted 03 April 2012 Abstract

Molecular modeling provides a mechanistic hypothesis at the molecular level of an unique extracelluar glutaminase free L-asparaginase from novel marine Actinomycetes.The physicochemical characteristics and the 3D models provide a framework

for the purification and characterization of Streptomyces radiopugnans MS1 L-Asparaginase.The Ramachandran plot for the

model was observed as 87.9 percentages of residues is in most favored region that indicate the model is reliable.The energy

value, instability index and RMSD (Root men square deviation) fluctuation of Carbon alpha back bone of the model was

computed that confirms the stability of the model protein. In the present study homology model of L-asparaginase from

Streptomyces radiopugnans MS1 was evaluated and compared it with other reported sources of the enzyme. Ligand binding

studies with both l-asparagine and l-glutamine using the L-asparaginase structure showed interesting results when compared

with E.coli and Erwinia L-asparaginase. The result indicates the possibility of Streptomyces radiopugnans MS1 protein being a

potential alternative to E.coli and Erwinia enzyme.

© 2011 Universal Research Publications. All rights reserved

Keywords: Molecular modeling, Actinomycetes, glutaminase free L-asparaginase, Ligand Binding and Ramachandran plot

Introduction

L-asparaginase is exploited in chemotherapy

schedule in the treatment of malignancies of the lymphoid

system, acute lymhoblastic leukemia and non-Hodgkin‟s

lymphoma [1]. Its anti-malignant activity is allied with the

possessions of exhausting the circulating pool of L-

asparagine by the asparaginase catalytic activity [2].

The use of L-asparaginase in anti-cancer therapy is

based on its ability to cleave L-asparagine, an amino acid

vital for malignant growth, to ammonia and L-aspartic acid in

serum and cerebrospinal fluid. Since malignant growths are incapable to produce endogenous L-asparagine,

malnourishment for this amino acid leads to fatality of these

cells [3].

Based on this, L-asparaginase has also been included

in most contemporary, multi-agent regimens for adult acute

lymphoblastic leukemia.

Broome, while working in Kidd‟s laboratory,

succeeded in 1961 in demonstrating that the antilymphoma

activity in guinea pig sera was due to L-asparaginase [4].

Since production of sufficient quantities of the enzyme from

the guinea pig serum is difficult, the scientific community

was in search for alternative methodologies.

It was a breakthrough when Mashburn and Wriston

in 1964 [5] reported the purification of Escherichia coli L-

asparaginase and demonstrated that its tumoricidal activity

was similar to that of guinea pig sera proving a practical base

for large-scale production of this enzyme for pre-clinical and

clinical studies. Though several Lasparaginases of bacterial

origin have been developed and their potential usage in clinical trials have been studied to prevent the progress of L-

asparagine-dependent tumors, mainly lymphosarcomas, the

success hitherto has been rather limited, and most of the

treatments must be interrupted due to severe side effects and

immunological reactions in the patients.

1. Literature reports indicated that the enzyme

biochemical and kinetic properties vary with the

genetic nature of the microbial strain analyzed [6].

Available online at http://www.urpjournals.com

International Journal of Research in Biotechnology and Biochemistry

Universal Research Publications. All rights reserved

2 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

For example, Erwinia L-asparaginase exhibited

less allergic reactions compared to the E. coli

enzyme. However, Erwinia asparaginase had a

shorter half-life than E. coli [7], suggesting the

need to discover new L-asparaginases that are

serologically different but have similar therapeutic effects.

This may have need of the screening of Marine

samples from various sources for isolation of potential

microbes, which have the ability to produce the desired

enzyme.

In this perspective, a bacterial strain belonging to the

Streptomyces radiopugnans MS1 species has been isolated in

our laboratory. Our preliminary investigation indicated that

this strain has the potential to produce extracellular L-

asparaginase. Keeping this in view, in this investigation, we

have studied the antineoplastic activity of this extracellular L-

asparaginase and report that this enzyme has potential for being used as antileukemia drug.

A convenient way to study the function, structure

and mechanism of a gene is to identify homologues

(evolutionary relationships) in model organisms.It is

interesting to identify homologues of L-Asparaginase in

Streptomyces sps. Dynamic programming based alignment

tools such as BLAST and FASTA have been widely used to

provide evidence for homology by matching a new sequence

against a database of previously annotated sequences.

However, these approaches can only detect homologous

proteins that exhibit significant sequence similarity. The availability of web based tools and server

provides an excellent opportunity to characterize the

physiochemical properties of Streptomyces radiopugnans

MS1 L-Asparaginase as well as their primary, secondary and

three dimentional structural properties.The purpose of this

study was primarily to report structural analysis and

characterization of Streptomyces radiopugnans MS1 L-

Asparaginase.

Methodology

Sequence and structure alignment

L-asparaginase gene from Streptomyces radiopugnans MS1 (GenBank: AEC45572 & DBsource

accession no is JF799106) was used as a source sequence,

sequence alignments of its amino acid sequence against

Protein Data Bank [8] were performed by means of the

BLAST algorithm (the used default Blast parameters were: E

cut-off = 10, mask low complexity = yes).More than 70

Sequence alignment shows that the target and the template

(share 31% of sequence identity).

Because protein structures are more conserved than

DNA sequences, detectable levels of sequence similarity

usually imply significant structural similarity. Based on the

significant e-value and alignment among the investigated templates, Streptomyces sps L-asparaginase was selected as a

template to build a model for Streptomyces radiopugnans

MS1 L-Asparaginase. The amino acid sequence of the target

protein, (Streptomyces radiopugnans MS1 L-asparaginase)

was retrieved from NCBI database, a centralized resource

database which provides taxonomic, physicochemical and

molecular information (GenBank: AEC45572 & DB source

accession no is JF799106) and is composed of 322 residues.

The sequence was aligned with all reported sources of L-

asparaginase such as Streptomyces pristinaespiralis ATCC 25486 (NCBI Reference Sequence: ZP_06910947 & DB

source REFSEQ: accession NZ_CM000950) , Strepto-

myces albus J1074 (NCBI Reference Sequence: ZP_

06591693 & DBsource REFSEQ: accession NZ_ DS999645),

Streptomyces sp. SPB78 (NCBI Reference Sequence:

ZP_07273143 & NZ_GG657742 and Strepto-myces sp. AA4

(NCBI Reference Sequence: ZP_ 07283361 & DBsource

REFSEQ: accession NZ_ GG657746).Using “ClustalW” for

determination of homology or similarity [9,10 & 11 ]

(http://www.ebi.ac.uk/Tools/clustalw).

Characterization of Protein Sequenced

Domain region was one of the most important data in protein sequence for identification of domain region from

the sequence “BLOCK” (http://blocks.fhcrc.org) server [12]

and “SMART” tools [13] were used. Other tool like “PRED

TMBB” tool was used to analyze the amino acid positions

where there is in or out position [14]

(http://bioinformatics.biol.uoa.gr/PRED-TMBB).

Prediction of Protein Structure

In first step, secondary structure of the protein was

predicted through “SOPMA” program [15] (Self-Optimized

Prediction Method) and the program determined the role of

individual amino acid forbuilding the secondary structure with their positions [16,17,18,19,20]

(http://npsapbil.ibcp.fr/cgibin/npsaautomat.pl?page=/NPS

A/npsa_sopma.html) and “TMpred”were used to predict

membrane-spanning regions of the protein and their

orientation. (http://www.ch.embnet.org/software/TMPRED -

form.html). In second step, the protein structure was

predicted by homology modeling with two different software

and server “Swiss model” [21, 22 & 23] and “Phyre2”

(http://swissmodel.expasy.org/workspace/index.php ,

http://www.sbg.bio.ic.ac.uk/phyre/index.cgi ).

Characterization of Structure The amino acid composition of the L-asparaginase was

computed using the PEPSTATS analysis toll [24]

( http://mobyle.pasteur.fr/data/jobs/pepstats ).The physico

chemical parameters such as the molecular weight, isoelectric

point(pI), extinction coefficient,half –life,aliphatic index,

amino acid property, instability index and Grand Average

Hydropathy (GRAVY) were calculated using the ProtParam

tool of the Expasy proteomics server.Secondary structure

elements predication was performed using the network

protein sequence analysis server and the Secondary Structural

Content Prediction (SSCP) server [25 & 26].The 3-D models

of L-asparaginase was constructed using the protein structure homology model building program SWISS-MODEL with

energy minimization parameters .The modeled tertiary

structure [27,28 ] were built on the basis of the sequence

3 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

“Figure:1, CLUSTAL 2.1 multiple sequence alignment of

L-Asparaginase Sequences.Single fully conserved residues

are represented by (*), deleted region(-),conservation of

strong and weak groups is denoted by (:) and (.),

respectively”.

homology with the high –resolution crystal structure of the

Streptomyces asparaginase enzymes.

Evaluation and validation of refined model

To obtain an accurate homology model, it is very

important that appropriate steps are built into the process to

assess the quality of the model. Therefore, accuracy of the

predicted models were subjected through a series of tests.

Stereochemical quality were evaluated using Ramachandran

plots obtained from the RAMPAGE server [29] & protein

structure validation suite (PSVS) and amino acid

environment was assessed using PROCHECK [30,31]

,WHAT_CHECK ,PROVE Verify 3D [32] and Errat [33] from the UCLA-DOE server [34] (http://nihserver.mbi.ucla.

edu). “ProSA” (https://prosa.services.came.sbg.ac.at/ prosa.

php ). While ProSA is a tool widely used to check 3D models

of protein structures for potential errors [35] and the

WHATIF server (http://swift.cmbi.ru.nl/servers/html/index.

html) validated structure scale and symmetry, atom

coordination, nomenclature, geometric, accessibility,

bumping, 3-D database, B-factor and hydrogen bond [36].

Comparison of protein models:

“Topmatch” tool was used for determination of the

superposition and structure alignment [37].Given a pair of protein structures, “TopMatch” calculates a list of alignments

ordered by structural similarity. The corresponding

superposition can be explored in a 3D molecule viewer.

(http://topmatch.services.came.sbg.ac.at/topmatch.html ).

“Figure: 2, The graphical view of the amino acids

position.A,B & C”.

Ligand binding sites analysis

The top ranking model of L-Asparginase has been

submitted to the 3DLigandSite server (http://www.sbg.bio.

ic.ac.uk/3dligandsite) [38] to predict potential binding sites.

Docking of substrates to L-Asparaginase

The L-Asparaginase normal substrate was L-Asparagine.In order to validate the active site architecture of

the Streptomyces radiopugnans MS1 L-asparaginase model

and examine its possible mode of interaction with the ligand,

the L-Asparagine substrate was docked within the

asparaginase homology model using the HEX v.6.3 [39]

docking environment at its default parameters. Hex is a tool

for macromolecule docking and it can superpose pairs of

molecules using only knowledge of their 3D shapes. Further,

it is one of the few docking tools having in built graphic

viewer [40, 41, 42 & 43]. This tool has been used in some

earlier studies demonstrating ligand- protein interaction. The

approach was to use blind docking since it has been recommended for acquiring good results in prediction of

substrate binding site [44] Correlation type and post-

processing output for receptor and ligand were kept based on

shape, electrostatic potential and minimization of molecular

mechanics (MM). Docking was carried out at full rotation

4 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

“Figure:3 Secondary structure of the l-asparaginase sequence through SOPMA”.

allowing full flexibility for the ligand while keeping receptor

position fixed in space [45].Docking parameters involved

Fourier transformation, steric scan, final search for ligand

binding site and refinement of the complex. Visualization

The results of the computational structure of Streptomyces

radiopugnans MS1 L-Asparaginase and its characterization

and validation by the various softwares and servers were

visualized by using softwares like “Phyre2” (http://www.sbg.

bio.ic.ac.uk /phyre2) “PyMOL” (http://pymol. sourceforge. net/) and “Chimera” (http://www.cgl.ucsf. edu/chim era/).

Result

Characterization of Protein Sequence

The multiple alignment result showed significant

homology was found with Streptomyces radiopugnans MS1

and homology with Streptomyces sps .The multiple

alignment score was showed that they are very close to each

other ranging from 50 to 79 % as shown in Table 1.Figure 1

shows the result of the multiple sequence alignment of the

mentioned organisms using CLUSTALW program.The

multiple sequence alignment also reveled that there are

stretches of amino acids that are exclusive to Streptomyces sps L-asparaginase.

Block analysis indicated that there were six blocks

found which was of L-asparaginase II family at positions like

13-52, 53-82,122-166,206-239,246-296 and 302-322 its

block sequence were PVLAEVVRSGFTEGHHRGSLVLL A

ADGSVDLALGDPAAP, FPRSSNKPMQAAAIL RAGL E

LSGERLALAA, AEAYLAAGRVREPLTMNCSGKH A A

MLAVCVRNGWDTATYLDPAHP, AFRAFVTAEPG SA

ERRVADAMRAHPEYVAGTRRP, EVPGTLSKMGAE A

VQAVALADGRALAFKIDDGSTRALGPVLARALELLGV

D & RIGRAPLLGGAEEVGRIRAAF respectively. Further analysis with “SMART” tool indicated that

there was one domain present in the sequence between 15 and

321 with an E-value 4.70e-102. The identification of conserved

domains from the sequence was done through “Motif Scan”.

The results indicate that amino acids from 15-321were highly

conserved for L-asparaginase II family.

“PRED TMBB” tool was used to analyze the amino acid

positions showed that 65-75 and 90-102 amino acids were in

Trans membrane, where 1-64 and 103-322 amino acid were

in inner membrane and 76-89 amino acid were

“Figure:4 TM pred output for L-Asparaginase enzyme.”

present in outer membrane (Figure 2). Sequence scored a value of 2.835, which is lower than the threshold value of

2.965. The difference between the value and the threshold

indicates the possibility of the protein being an outer

membrane protein.

Prediction of Protein Structure

The result obtained from “SOPMA” is presented in

the form of graphics (Figure 3). The tool described that about

39.75 % of amino acids presented in Alpha helix, 10.25% of

amino acids in beta turn, 36.02% of amino acids in random

coil, 13.98% of amino acids in extended strand and rest of all

amino acids in bridge and turn (Figure-3). “TMpred” suggestions are purely speculative and

should be used with extreme caution since they are based on

the assumption that all transmembrane helices have been

found.In most cases, the Correspondence Table-3 shown or

the prediction plot (Figure-4) that is also created should be

used for the topology assignment of unknown proteins.

2 possible models considered, only significant TM-

segments used

-----> STRONGLY preferred model: N-terminus outside

1 strong transmembrane helices, total score: 1238

# from to length score orientation 1 183 204 (22) 1238 o-i

------> alternative model 1 strong transmembrane helices, total score: 922

# from to length score orientation1 185 204 (20) 922 i-o

The homology model was builted from sequence of

L-asparaginase from Streptomyces radiopugnans MS1and as

per multiple alignment result Strptomyces l-asparaginase

used as a template structure. The homology modelling was

done through the swissmodel server and phyre2 were founded

as the best server among of all others software or server. The

predicted structure quality was good through different

software as compared with others.Figure 5 & 6 showed the

structure obtained through “swiss model workspace server” and “Phyre 2”respectively.

5 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

“Figure: 5, Swiss model workspace reports.”

“Figure :6, Secondary structure and disorder prediction by

Phyre2”.

Characterization of Structure:

The amino acid composition of the L-asparaginase

was showed in table table: 3 (A) and (B).The sequence of L-

Asparaginase was analyzed by the computer program

Protparam in order to find the physical and chemical

properties. (Table: 4)

Evaluation and validation of refined model

The model generated by the above method was

subjected to validation using the following softwares: I)

Ramachandran Plot using RAMPAGE server: The

Ramachandran plot for the modeled structure showed 87.9%

of the residues in the most favored region, 6.4 % in the additional allowed region, and 5.8% in the generally allowed

region and no residues in the unfavorable region (Figure-7)

and Ramachandran Plot Statistics from Richardson's lab

(Figure-8).

“ProSA” tool graph showed all over model quality

of the structure and the location of the z-score for the

structure. The value, -1.39, was in the range of native

conformation. It also represents the point of the structure

which was within a range which was determined by X-ray

and NMR studies (Figure 9 A & B).

The root mean square deviation (RMSD) score was calculated using the sequence identity and gaps in the

alignment displaying RMSD for bond anglees 4.6o, bond

“Figure :7, Ramachandran plot values showing number of

residues in favoured, allowed and outlier region”.

“Figure:8, Residue Plot of Ramachandran anlysis(based on

data from Richardson Lab's Molprobity”.

lengths 0.026 Å ,RMS Z-score : 1.951 and RMS-deviation in

bond distances: 0.026.zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz

Validation report of WHATIF server described that all over

6 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

“Figure:9 A, Overall model quality--“ProSA” tool graph”.

“Figure:9 B, Local model quality-“ProSA” tool graph”.

“Figure: 10, Errat plot for Streptomyces radiopugnans MS1 L-Asparaginase model. Black bars show the misfolded region

located distantly from the active site, gray bars demonstrate

the error region between 95% and 99%, white bars indicate

the region having less error rate for protein folding.

Expressed as the percentage of the protein for which the

calculated error value falls below the 95% rejection limit,

good high resolution structure generally produce values

around 95% or higher. For lower resolution (2.5 to 3A) the

average over all quality factor is around 91%”.

the structure was alright with negligible error. The tool

validated structure scale and symmetry, atom coordination,

nomenclature, geometry, accessibility, bumping, 3-D

database, B-factor and hydrogen bond. Mostly in all

parameters reports were alright and Z score was obtained

within range. Errat plot assesses the arrangement of different types

of atoms with respect to each other in protein models (Fig.

10). Errat is a sensitive technique, which is good for

identifying incorrectly-folded regions in preliminary protein

models. ERRAT is a so-called „„overall quality factor‟‟ for

non-bonded atomic interactions, and higher scores mean

higher quality22. In the current case, the ERRAT score for

the model is 25.641.

L-asparaginase was validated with Phyre2 177

residues (55% of your sequence) have been modelled with

96.6% confidence by the single highest scoring template

(Figuer 11).

Comparison of the structure of L-asparaginase From the time when the alignment results of

Streptomyces radiopugnans MS1 Lasparaginase showed

homology to Streptomyces pristinaespiralis ATCC 25486

[46] L-asparaginase II. The structure of L-asparaginase was

superposed with the Streptomyces pristinaespiralis ATCC

25486 (Sippl 2008). L-asparaginase II, which showed that in

10 residues pairs that were structurally equivalent the

identities of the overall structure were around 84% (Figure

12).

Binding site analysis of L-Asparaginase Once final model was build and validated, the

possible binding sites of L-Asparaginase were searched using

Phyre2 server [47] .Eight different Ligand cluster were

identified and predicted binding aminoacid were Ala (158), Thr (159),Met (254),Gly (255),Ala (256).Table:5 (A & B)

,Figure 13 and Hetrogens present in predicated Binding site

(SUC and CFX) Table (5 C).

Docking For docking, the ligand structure were obtained from the

PubChem database.33 In order to investigate the substrates

binding with the enzyme, we attempted to dock L-Glutamine

and L-Asparagine and to L-Asparaginase enzyme model of

Escherichia coli str. K-12 substr. MG1655, Pectobacterium

atrosepticum SCRI1043 and Streptomyces pristinaespiralis

ATCC 25486. The top docking solutions of 3000 inter action

results for each ligand was selected. This result confirms that

the most preferred substrate for Streptomyces radiopugnans MS1 is the L-Asparagine. Since its binding energy is the

smallest one (Table-6).

DISCUSSION In the current study, the L-asparaginase protein was

evaluated from gene to protein. The homology modelling was

done through the http://www.ebi.ac.uk/Tools/clustalw, which

was found as the best.

Software among of all other software or server. The

7 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

“Figure:11, Model validated by Phyre2”.

“Figure:12, Comparison of protein models by Topmatch

tool”.

structure obtained through computation was validated

through Phyre2, ProSA, Verfy 3D and Errat.The structure

was naturally stable with low energy value. In WHATIF

report, validation of the structure of L-asparaginase was OK.

In structure scale and symmetry, atom coordination,

nomenclature, geometric, accessibility, bumping, 3-D

database, B-factor and hydrogen bond, mostly in all parameters reports were ok with getting the Z score within

the range. The predicted structure was superposing with

reported sources and up to 79% homology was found with

Strptomyces L-Asparaginase. The result showed that the

predicated structure might be a favorable protein for human

for the treatment of ALL.

“Figure:13, Structural view of predicated potential binding

sites by 3DLigandSite server”.

“Figure:14, Validate the active site architecture of the

Streptomyces radiopugnans MS1 L-asparaginase model with

L-Asparagine”.

The ligand efficiency was one of the major points in structure

characterization. The model which was obtained from

computational method also gives a favorable binding

efficiency with the L-asparagine. The active site was found

different eight places on the model, among these; one was

ideally efficient against the substrate with gives lowest

energy value for docking. The residual of the active site where most of similar with Streptomyces sps active site

residual which was obtained from crystallography methods.

The structure showed high efficiency towards the lasparagine

as well as no efficiency against L-glutamine, so may be

L-asparaginase from Streptomyces radiopugnans MS1

will be a novel source for treatment for ALL as its have no

8 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

Table 1: CLUSTAL 2.1 Multiple Sequence Alignments Score of L-Asparaginase enzyme

SeqA Name Length SeqB Name Length Score

1 Seq1 322 2 Seq2 324 79

1 Seq1 322 3 Seq3 334 74

1 Seq1 322 4 Seq4 321 74

1 Seq1 322 5 Seq5 314 50

2 Seq2 324 3 Seq3 334 73

2 Seq2 324 4 Seq4 321 71

2 Seq2 324 5 Seq5 314 53

3 Seq3 334 4 Seq4 321 72

3 Seq3 334 5 Seq5 314 51

4 Seq4 321 5 Seq5 314 51

Note: Seq1: Streptomyces radiopugnans MS1,Seq2 : Streptomyces pristinaespiralis ATCC 25486,

Seq3: Streptomyces albus J1074 Seq4: Streptomyces sp. SPB78,Seq5 : Streptomyces sp. AA4.

Table 2: Here is shown, which of the inside->outside helices correspond to which of the

outside->inside helices.Helices shown in brackets are considered insignificant.

Inside -> outside | outside -> inside

(30 - 53 (24) 175) | (30 - 54 (25) 303 +)

185 - 204 (20) 922 | 183- 204 (22) 1238 ++

A"+"-symbol indicates a preference of this orientation.

A "++"-symbol indicates a strong preference of this orientation.

Table 3(A): The amino acid composition of the L-asparaginase was computed using the PEPSTATS.

Residue Number Mole% DayhoffStat

A = Ala 58 18.012 2.094

C = Cys 3 0.932 0.321

D = Asp 17 5.28 0.96

E = Glu 19 5.901 0.983

F = Phe 7 2.174 0.604

G = Gly 31 9.627 1.146

H = His 8 2.484 1.242

I = Ile 5 1.553 0.345

K = Lys 4 1.242 0.188

L = Leu 40 12.422 1.679

M = Met 10 3.106 1.827

N = Asn 5 1.553 0.361

P = Pro 23 7.143 1.374

Q = Gln 4 1.242 0.319

R = Arg 25 7.764 1.584

S = Ser 16 4.969 0.71

T = Thr 14 4.348 0.713

V = Val 28 8.696 1.318

W = Trp 2 0.621 0.478

Y = Tyr 3 0.932 0.274

9 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

Table 3(B): The amino acid composition of the L-asparaginase was computed using the PEPSTATS.

Property Residues Number Mole %

Tiny (A+C+G+S+T) 122 37.88

Small (A+B+C+D+G+N+P+S+T+V) 195 60.559

Aliphatic (A+I+L+V) 131 40.683

Aromatic (F+H+W+Y) 20 6.211

Non-polar (A+C+F+G+I+L+M+P+V+W+Y) 210 65.217

Polar (D+E+H+K+N+Q+R+S+T+Z) 112 34.783

Charged (B+D+E+H+K+R+Z) 73 22.671

Basic (H+K+R) 37 11.491

Acidic (B+D+E+Z) 36 11.18

Table 4: Physicochemical and structural data of Streptomyces radiopugnans MS1 of L-Asparaginase extracted from ProtParam

tool of the Expasy proteomics server”.

S.No Physicochemical Character Data

1 Aminoacid Length: 322

2 Average Theoretical pI/Mw: 5.64 / 33319.27

3 Monoisotonic Theoretical pI/Mw: 5.64 / 33340.28

4 Charge: -3

5 Negatively charged residues (Asp + Glu): 36

6 Positively charged residues (Arg + Lys): 29

7 Abscent amino acid: B,Z,X

8 Common amino acid: A,L,G

9 The instability index (II): 27.55, protein as stable

10 Aliphatic index: 97.73

11 Total number of atoms: 4709

12 Formule: C1461H2370N428O437S13

13 Grand average of hydropathicity (GRAVY): 0.167

14 The estimated half-life 30 hours

15 A280 Molar Extinction Coefficients 15470 (reduced) 15595 (cystine bridges)

16 A280 Extinction Coefficients 1mg/ml 0.464 (reduced) 0.468 (cystine bridges)

17 Improbability of expression in inclusion bodies 0.586

Table 5A: Binding site analysis of L-Asparaginase.

Ligand Clusters Identified

Note prediction based on first cluster

MAMMOTH Scores

Cluster Ligands Structures Av min max

1 4 4 11.6 9.5 13.2

2 3 3 10.6 8.4 12.4

3 2 2 10.8 9.5 12.2

8 1 1 15.7 15.7 15.7

6 1 1 13.1 13.1 13.1

7 1 1 13.1 13.1 13.1

5 1 1 8.4 8.4 8.4

4 1 1 8.3 8.3 8.3

10 International Journal of Research in Biotechnology and Biochemistry 2012; 2(1) : 1-12

Table 5C: Heterogens present in Predicted Binding Site.

Heterogens present in Predicted Binding Site

Heterogen Count source structures

SUC 2 1l0g_A,2hds_A

CFX 2 1i2w_B,1ymx_B

Table 6: Substrate specificity through docking.

S.No Protein Source Ligand

Estimated Free

energy of

Binding

1 Streptomyces radiopugnans MS1 L-Asparagine -125.25

L-Glutamine -0.18

2 Streptomyces pristinaespiralis ATCC 25486 L-Asparagine -119.53

L-Glutamine -0.75

3 Escherichia coli str. K-12 substr. MG1655 L-Asparagine -118.17

L-Glutamine -105.18

4 Pectobacterium atrosepticum SCRI1043 L-Asparagine -117.37

L-Glutamine -66.46

L-glutaminase activity.

The wet lab data on the enzyme- substrate kinetics

were supported to docking data against l-asparagine [48].In

the present study, a homology model of Bacterial L-

asparaginase from Streptomyces radiopugnans MS1 was

obtained in order to provide a reliable model with which to

design new inhibitors and to investigate the treatment of ALL using novel protein sources. In the absence of the

experimental structure of Lasparaginase,it is believed that the

model presented through this work will be useful for further

studies on novel sources of Lasparaginase for treatments of

ALL.

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Source of support: Nil; Conflict of interest: None declared