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International Journal of Biological Macromolecules 47 (2010) 298303

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International Journal of Biological Macromolecules
j our nal homepage: www. el sevi er . com/ l ocat e/ i j bi omac
Homology modeling and docking study of xanthine oxidase
of Arthrobacter sp. XL26
R.G. Bodade
a
, S.D. Beedkar
a
, A.V. Manwar
b
, C.N. Khobragade
a,
a
Biochemistry Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathawada University, Nanded, Maharashtra 431606, India
b
Department of Microbiology, Dyanopasak College, Parbhani, Maharashtra 431402, India
a r t i c l e i n f o
Article history:
Received 24 March 2010
Received in revised form 10 April 2010
Accepted 12 April 2010
Available online 18 April 2010
Keywords:
Homology modeling
Arthrobacter sp. XL26 XO (xodB protein)
Docking
a b s t r a c t
Hyperuricemia is a condition of defective purine metabolism characterized with elevated serum uric
acid (UA) level that further leads to gout and gouty nephrolithiasis disorders. Gout is a world wide
distributed rheumatic disease comprises 1% of the total population and still is in increasing state. One of
the factors contributing to overproduction of UA is the hydroxylation of xanthine catalyzed by xanthine
oxidase (XO). In the present study, 3D modeling of Arthrobacter sp. XL26 (xodB) protein was performed
by comparative modeling approach using Rhodobacter capsulatus XDH (PDB ID: 2W3sF) as template in
SWISS-MODEL, Geno3D and MODELLER program server. The best model was selected based on overall
stereochemical quality (Procheck, PROSA, GenThreader), energy minimized, rened and used for active
site characterization in BioMed CAChe workspace. The enzymeinhibitor interaction was studied by
docking to screenthe possible inhibitors andapplicationof model indesignanddevelopment of anti-gout
agents.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Xanthineoxidoreductase (XOR) is a complex metallo-
avoprotein that catalyzes the oxidative hydroxylation of purines,
pyrimidines, pterins and aldehyde substrates. It is also a key
enzyme of purine pathway and catalyzes the conversion of
hypoxanthine to xanthine and xanthine to uric acid (UA) with
concomitant production of hydrogen peroxide and superoxide
anions. It exhibits in two alternate forms of a same gene product as
xanthine dehydrogenase (XDH, EC 1.1.1.204) and xanthine oxidase
(XO, EC 1.2.3.2) [1,2]. Structurally the enzyme is a homodimer of
molecular weight 290,000andeachsubunit of the enzyme contains
one molybdo-pterin(MO-Pt) cofactor, two distinct ironsulfur cen-
ters (2Fe2S) and one avin adenine dinucleotide cofactor (FAD)
[3]. The accumulation of UA is known to cause hyperuricaemia,
gout and UA nephrolithiasis and hence, inhibitors of UA formation
could be useful as therapeutic agents for theses disease [4]. In
addition a large amount of superoxide anion generation by XO has
been involved in the pathogenesis of inammation, mutagenesis,
cancer, ageing andischemic reperfusioninjury, therefore inhibitors
of the generation and radical scavengers of superoxide anion are
also useful for prevention of the oxidative damaged induced
diseases [5]. Moreover, gout is also associated with other diseases
like hypertension, hyperlipidation, diabetes mellitus, obesity and

Corresponding author.
E-mail address: raginibodade@rediffmail.com (C.N. Khobragade).
cardiovascular diseases [6]. The enzyme amino acid (1330 AA)
sequence is highly homologous among the rat, mouse and human
enzymes with about 90%identity [7]. Awell characterized prokary-
otic XDH with similar activity to the eukaryotic counterparts is
the enzyme isolated from the phototrophic purple bacterium
Rhodobacter capsulatus. The amino acid sequence of RcXDH has
a high degree of similarity to eukaryotic XDH/XO. Because of
the high structural and sequence similarity of RcXDH and bXDH
especially in the active site, the bacterial enzyme is a good model
system for studying the mechanism and design of the new, more
effective clinical inhibitors [8]. Allopurinol is the most common
clinically used XO inhibitor prescribed for the treatment of gout.
However its use sometimes limited by hypersensitivity problems,
StevensJohnson syndrome, renal toxicity, fetal liver necrosis and
because of its poor scavenging activities [9]. Although, newas well
as previously known compounds have recently been discovered
to inhibit XDH/XO activity, a clinically effective inhibitor has not
been developed to treat hyperuricemia after allopurinol. Recently
febuxostat has been approved by FAD for gout treatment [10]. XO
of Arthrobacter sp. XL26 has been puried and well characterized
[11]. The enzyme revealed to has a MW of 260,000 and consists
of heterogeneous subunits xodA and xodB that showed homology
with Acinetobater baumannii ATCC 17978 XDH. Two-dimensional
structure models indicate its (2Fe2S) and FAD binding domains
locate in xodA, whereas Mo-binding and a/b hammer head-domain
of XDH seat in xodB. The crystal structure data (1FiQ) revealed
direct substrate binding and oxidation reaction, at the MO-Pt
center or MO ion of the MOCO [12].
0141-8130/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2010.04.002
R.G. Bodade et al. / International Journal of Biological Macromolecules 47 (2010) 298303 299
Therefore, knowledge of the 3Dstructure of a proteinis essential
to understand how protein performs its function. Protein struc-
tures can be determined at high resolution by either experimental
methods such as X-ray crystallography and nuclear magnetic res-
onance (NMR) or computational analysis (i.e. using bioinformatics
tools) [13]. In the absence of crystallographic structure, a variety
of advanced homology modeling methods have been developed,
which can provide reliable models of proteins that share 30% or
more sequence identity with a known structure [14]. Such models
also have proven useful during drug design projects and allowed
the taking of key decisions incompoundoptimizationandchemical
systems [15].
In continuation of our research work [1618] to screen novel
XOR inhibitors, we puried and characterized the XOfromisolated
Arthrobacter strain (data unpublished). In the present study, the
homology model of heterogeneous subunit from Arthrobacter sp.
XL26 (xodB) model has been generated by a comparative modeling
approach using R. capsulatus XDH (PDB ID: 2W3sF) chain as tem-
plate. The constructed model was further validated by structure
analysis tools like PROSA and PROCHECK to analyze its structural
integrity. The active site prediction and docking studies were per-
formed to conrm its application for inhibitor screening.
2. Methods
2.1. Software and hardware
Automatedhomologymodelingwas performedbyusingSWISS-
MODEL [19], Geno3D [20] and MODELLER 7v7 (http://salilab.org).
Model was evaluated by PROCHECK[21], PROSA[22] and Verify-3D
[23]. Docking studies with BioMed CAChe workspace module was
carried out [24]. Interactive visualization and analysis of molecular
structures was carried out in Swiss PDB viewer [25].
2.2. Sequence retrieval alignment and homology modeling
The 3D model of heterogeneous subunit of XO (xodB)
from Arthrobacter sp. XL26 was built by homology modeling
based on high-resolution crystal structures of homologous pro-
teins. The complete amino acid sequence of the target protein
Arthrobacter sp. XL26 (xodB) was retrieved from NCBI protein
sequence database (Accession No. GI: 157058375) in FASTA for-
mat (http://www.ncbi.nlm.nih.gov/Proteins/). The NCBI Basic Local
Alignment Search Tool (BLAST), for the sequence similarities was
used for searching the crystal structures of the closest homologues
available in the Brookhaven Protein Data Bank (PDB). The results
yielded by NCBI BLAST revealed XDH (2W3sF) from R. capsulatus
(PDB ID: 2W3S) with a resolution of 2.6 as a suitable template
andwithanidentity score of 51%andE value 0.0. The coordinates of
crystal structure of XDH(2W3sF) fromR. capsulatus (PDBID: 2W3S)
were used as template to build the model by pair-wise sequence
alignment using Clustal W software [26]. GenThreader the fully
automatic fold recognition server was used for fold assignment in
target [27].
Homologymodelingof xodBwas performedbythree automated
homology modeling programs: SWISS-MODEL, Geno3D and MOD-
ELLER using single template downloaded from the Brookhaven
PDB (http://www.rcsb.org/pdb/) 2W3sF by comparative modeling
approach [28]. The steepest descent energy minimization using the
GROMOS 96 force eld was done to regularize the protein structure
geometry [29].
2.3. Model renement
The model was renedby performing anoptimize geometry cal-
culationinmechanics using augmented MM3 parameter at BioMed
CAChe/workspace module. CAChe molecular mechanics includes
energyterms for bondstretch, bondangle, dihedral angle, improper
torsion stretch, bond bend, van der waals electrostatics and hydro-
gen bond interactions. The rened model was again subjected
to energy minimization using GROMOS and evaluated for quality
assessment.
2.4. Validation of the 3D structure
The validation for structure models obtained from the different
software tools (SWISS-MODEL, Geno3D and MODELLER) was per-
formed by inspection of the Psi/Phi Ramachandran plot obtained
from PROCHECK analysis. Out of these, the model constructed by
SWISS-MODEL was nally chosen for further investigations as it
possessedthebest geometryandenergyprole. ThePROSAtest was
applied for nal model to check for energy criteria in comparison
with the potential of mean force derived froma large set of known
protein structures. Further, the compatibility of the model with its
sequence was measured by Verify-3D. The root mean square devia-
tion (RMSD) between the main-chain atom of model and template
was calculated by structural superimposition of template (2W3sF)
and predicted structure of xodB heterogeneous subunit for the reli-
ability of the model performed using combinatorial extension of
polypeptides.
2.5. Active site prediction
The active site prediction in the rened model was done by
automatic sequence alignment mode in BioMed CAChe workspace.
Active site was locatedintemplate rst (2W3sF) by selecting ligand
(xanthine), embedded ina 5shell of residues, water and HETs and
saved as active site pocket. Active site pocket is the surface of the
protein adjacent to the ligand and the surface that forms a pocket
around the ligand. The active site detection in the rened model
was donebyautomatic sequencealignment modeinBioMedCAChe
workspace. We aligned the sequences of the homology model with
the sequence of the homologous template (2W3sF) for which the
active site is known. We identiedthe active site residues inhomol-
ogy model from the alignment. The gaps appear in the homology
model and template was aligned in the sequences according to
the maximum scoring alignment using BLOSUM 50 substitution
matrix in the NeedlemhamWunsch alignment algorithm[3032].
The matching residues in the homology model were discovered by
match selection by choosing 2W3sF as the master sequence.
2.6. Docking ligand into homology model
The inhibitors were selected and rened for lowest energy
rotamer by molecular mechanics procedure at BioMed CAChe 6.1.
It was then docked over the homology model. CAChe automates
the docking of ligand into the active site using a genetic algorithm
with a fast, simplied potential of mean force (PMF) [33]. PMF
has been demonstrated to show a signicant correlation between
experimental binding afnities and its computed score for diverse
proteinligand complexes [3438].
3. Results and discussion
3.1. Homology modeling of xodB from Arthrobacter XL26
Homology modeling is usually the method of choice when a
clear relationship of homology between the sequence of target
protein and at least one known structure is found. This approach
would give reasonable results based on the assumption that the
tertiary structures of two proteins will be similar if their sequences
are related [39]. The absence of the three-dimensional structure
300 R.G. Bodade et al. / International Journal of Biological Macromolecules 47 (2010) 298303
Fig. 1. Sequence alignment between XO of Arthrobacter sp. XL26 (xodB) protein and Rhodobacter capsulatus XDH (2W3sF chain) in Clustal W.
Table 1
Possible template for 3D structure prediction of xodB protein.
Conf. Net score p-Value Pair E Solv E Aln score Aln Len Str Len Seq Len Alignment
CERT 355.668 6E35 1064.7 19.2 1924.9 753 1298 784 1v97A0
CERT 353.648 1E34 1078.9 18.8 1908.2 754 1291 784 2e3tA0
CERT 351.939 1E34 1099.4 24.5 1869.9 759 760 784 2w3sB0
CERT 338.834 3E33 1049.3 14.1 1831 746 761 784 1rm6A0
CERT 336.203 6E33 1034 13.1 1818 760 786 784 1t3qB0
CERT 330.582 2E32 947.4 17.5 1787 755 795 784 1ffvB0
CERT 321.011 2E31 1017.9 10.7 1726 757 804 784 1n62B0
CERT 272.214 2E26 810.8 6.3 1463 720 906 784 1dgjA0
CERT 269.7 3E26 862.9 7.6 1427 723 907 784 1vlbA0
CERT 188.687 4E18 547.6 14.9 972 409 420 784 3hrdA0
CERT 134.254 1E12 435.6 3.6 702 328 330 784 3hrdB0
CERT 114.465 1E10 201.2 25.5 792 442 450 784 2w3sA0
CERT 75.258 1E06 270.9 40.8 575 552 574 784 3c8yA0
CERT 64.843 0.00001 87.8 16.7 403 338 340 784 3i99A0
for Arthrobacter xanthine oxidase (XO) in PDB prompted us to
construct its homology model. The sequence of Arthrobacter sp.
xanthine oxidase was searched in NCBI site giving only two results
as Arthrobacter sp. XL26 (GI: 157092381) and Arthrobacter sp. XL26
(xodB) protein(GI: 157058375). The complete sequence of boththe
possible targets was downloaded in FASTA format and the protein
BLAST for each complete protein sequence was executed, a hit was
given <30% similarity with the target protein.
Among the XO/XDH with known three-dimensional structures,
the 2W3sF XDH of the R. capsulatus (PDB ID: 2W3S) showed the
highest sequence identity with xodB from Arthrobacter XL26. Pri-
mary sequence alignment showed that target (xodB) and template
(2W3sF) share 51% identity with E value 0.0 as compared to other
templates. At this level of sequence identity, it is good enough
to use crystallographic structure of 2W3sF from R. capsulatus as
a template, in order to obtain high quality alignment for struc-
ture prediction by homology modeling (Fig. 1). The neighboring
joining plot for template and target also showed the similar ori-
gin at equal distance (23.19), conrming the structural homology
(Fig. 2). GeneThreader was used to recognize the fold assignment
for 3Dstructure predictionof Arthrobacter sp. XO(xodB) giving best
template score for 2W3sF as per Table 1.
The three-dimensional structure provides valuable insight into
molecular function and also enables the analyses of its interac-
tions with suitable substrates or inhibitors. Three models were
constructed by different protein prediction tools: Geno3D, SWISS-
MODEL and MODELLER. The predicted models were checked for
psi and phi torsion angles using the Ramachandran plots. A com-
parison of the results obtained from the different software tools,
shows that one of the model generated by SWISS-MODEL is more
acceptable incomparisontothose generatedby Geno3D, andMOD-
ELLER. Like 2W3sF crystal structure, the xodB homology model is
predominantly -helical in nature with -sheets as shown in Fig. 3.
3.2. Validation of the predicted structure
The possible applications of protein models depend largely on
the quality of models. The model was evaluated by the tools avail-
able at SWISS-MODEL. The ANOLEA results represent the Y-axis
of the plot, the energy for each amino acid of the protein chain.
Negative energy values (in green) represents the favorable energy
environment whereas the values (in red) unfavorable energy envi-
ronment for a given amino acid. There are few amino acids lying
in an unfavorable energy environment, which can be ignored in
overall consideration of the model structure. GROMOS has rep-
resented the Y-axis of the plot, showing energy for each amino
acid of the protein chain in the form of red (unfavorable region)
Fig. 2. Neighboring joining plot of Target (xodB) and Template (2W3sF) sequence.
R.G. Bodade et al. / International Journal of Biological Macromolecules 47 (2010) 298303 301
Fig. 3. Rened structure of homology model of xodB. The -helix is represented by
dark pink spirals, -sheet by yellowarrows and white are turns. (For interpretation
of the references to color in this gure legend, the reader is referred to the web
version of the article.)
or green (favorable region) colorization. The force led energy for
the overall structure is 1045.90(before renement) and1072.16
(after renement), conrm the homology model experimented.
Qumean score is a composite score consisting of a linear combi-
nation of six terms, which helps to estimate the quality of protein
structure model, in the form of a total Qumean score value. The
model Qumean score obtained for the model (0.71) is within the
reliability (01). The overall stereochemical quality of the model
was assessed by Procheck. The Ramachandran plot showed 94.1%
residues in most favorable region, 4.9% in allowed region, 1.1% in
additional and disallowed region as compared to 2W3sF template
(94%, 5.0% and 0.9%), respectively. The results revealed that major-
ity of the amino acids are in a phipsi distribution consistent with
right handed -helix and reliable to be good quality model (Fig. 4).
Fig. 4. Ramachandran plot of xodB homology model.
Fig. 5. PROSA energy plot calculated for the xodB homology model.
The G-factors, indicating the quality of covalent and bond angle
distance, were 0.26

for dihedrals, 0.20

for covalent, and over-


all 0.06

. The overall main-chain and side-chain parameters, as


evaluated by PROCHECK, are all very favorable. The Ramachandran
plot characteristic and G-factors conrm the quality of predicted
model.
In order to investigate whether the interaction energy of each
residue with the remainder of the protein is negative, a second test
was done to apply energy criteria using Prosa II energy plot. The
Prosa analysis of the model showed maximum residues to have
negative interaction energy with very few residues displayed pos-
itive interaction energy as shown in Fig. 5.
A nal test is the packing quality of each residue as assessed by
the Verify-3D program that represents the prole obtained with
respect to the residues. The compatibility score above zero in the
Verify-3D graph corresponds to acceptability side-chain environ-
ments (Fig. 6). In Verify-3D the vertical axis represents the average
3D1D protein score for each residue in a 21 residue sliding win-
dow helps to further validating the model (0.69) and score found
to be near to the template (0.72).
The quality of the model was also assessed by comparing the
predicted structures to the experimentally showed structure via
Fig. 6. The 3D proles veried results of xodB model, where residues with positive
compatibility score are reasonably folded.
302 R.G. Bodade et al. / International Journal of Biological Macromolecules 47 (2010) 298303
Fig. 7. Superimposed backbones of xodB homology model (blue) and template
2W3sF (pink). (For interpretation of the references to color in this gure legend,
the reader is referred to the web version of the article.)
superimposition and atoms RMSD assessment (Fig. 7). Conse-
quently superimposition of the template with homology model
was executedby combinatorial extensionof polypeptides. The RMS
deviation of C trace between homology structure and template
was 0.33 support that generated model is reasonably good and
quite similar to template. Hence, the nal model, which proved to
be well validated in terms of geometry and energy proles, sug-
gests that the model is good enough to be a starting point for our
next phase of docking studies.
3.3. Active site prediction and docking study
The proteinligand complexes give greater insights in structure
based drug design, so a proteinligand complex was developed.
It gives a more detailed and accurate picture of the interactions
and structural complementarities between the ligand and the
active site. The active site pocket of template revealed that the
ligand is highly embedded in hydrogen bond donor region of
the protein. The proteinligand complex in generated homol-
ogy model is developed by sequence alignment and match
Fig. 8. Active site (pink) for the xodB homology model. (For interpretation of the
references to color in this gure legend, the reader is referred to the web version of
the article.)
selection procedures. Assuming that the ligand binding modes
are similar in the target and the template protein structure,
the coordinates of ligand was transferred from 2W3sF crystal
structure. Comparative active site analysis of template (2W3sF)
and homology model (xodB) shows highly conserved residues
Glu232 (in model Glu245), Leu 303(Leu 316), Pro306(Pro319),
Arg310(Arg323), Ala340(A353), Phe344(Phe357), Ser458(Ser456),
Phe459(Phe457), Thr460(Asn458), Leu461(Ala459),
Leu464(Tyr462), Ala526(Ala524), Ala528(Ala526), Ala529(Ala527),
Ser530(Ser528), Glu730(Glu733), MO778(MO778). The active site
residues in homology model Asn458, Ala459 and Tyr462 are dif-
ferent from those residues involved in the active site of template
(2W3sF). The catalytic active site contains the MO (molybdenum
cofactor), which is conserved in both models, this conrms its
importance in biding with the inhibitor. Finally, docking study
of the model was performed in order to elucidate its structural
and functional relevance in terms of inhibitor binding. Allopurinol
and avone, the well-known inhibitors, were successively docked
on to the active site of homology model. Fig. 8 shows docking of
inhibitor on to the active site where the Ser456, Phe457, Asn458,
Ala526, Ala527 and MO778 are directly involved in biding with the
inhibitors. Table 2 shows the results of the docking experiments in
Fig. 9. Docking of inhibitors on active site of xodB homology model.
R.G. Bodade et al. / International Journal of Biological Macromolecules 47 (2010) 298303 303
Table 2
Dock score of inhibitors.
Inhibitor/substrate Dock score
Xanthine 95.471
Flavone 82.224
Allopurinol 84.520
terms of calculated free energy of binding. The substrate xanthine
as ligand gives highest score (95.471), conrms its tight binding
with the template/target. The inhibitor avone and allopurinol
also gives good score 82.224 and 84.520, respectively, conrms
the model has application in drug discovery (Fig. 9).
4. Conclusion
In the present work the 3D model of xodB was constructed in
order to accomplish its molecular modeling and docking studies.
The model was validated and further used for docking analysis
with some well-known inhibitors viz. avone and allopurinol. The
resulting docking solutions were analyzed for binding pattern and
conformational analysis study. Further work is concentrated on
screening of novel avones using the same model to make a major
impact on anti-gout chemotherapy.
Acknowledgment
The authors are thankful to Head, School of Life Sciences, SRTM
University, Nanded, for providing necessary facilities.
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