nst Life Sciences and Bioinformatics ISSN: 0974-9179

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Research Paper

In-Silico Modeling and Investigation of ATP Binding Pocket of

An Algal Oil Producing Enzyme

Raghunath Satpathy*, Rajesh ku.Guru, Rashmi R Behera and Aparajita P

Department of Biotechnology, MIRC LAB, MITS Engineering College, Rayagada, Odisha, India – 765 017.

(*Corresponding Author E-mail: rnsatpathy@gmail.com)

(Received March 11, 2010; Revised and Accepted March 31, 2010)

ABSTRACT
Acetyl-CoA carboxyl transfersae (ACCase) is the key enzyme for the synthesis of oils in algae. The
particular enzyme catalyses the reaction to form the Malonyl -CoA by binding with the substrate like ATP
and Coenzyme A. The present work is an in-silico approach which deals with the Homology based
modeling and identification of ATP binding residues of the enzyme ACCase of the biofuel producing algae
Cyanidium caldarium. MODELLER program has been used for 3D modeling. The reliability of the model
was verified on basis of PROCHEK, ERRAT, and, DOPE result. Then prediction of binding pocket and
docking study show that the residues ILE 110, ARG113, LEU 296 are responsible for binding to ATP. The
structural motif analysis for the protein sequence by EMBOSS show that Prosite motif present in the
sequence is AGRR (110-113), which are in an agreement with previous findings. The stability of the
docked structure is also checked by energy minimization by Chimera.

Key words: Homology modeling; algae; docking; biofuel; binding site.

INTRODUCTION
The development of alternative energy sources such as
biofuels are one of the most exciting and challenging
area. So the effort has been put to produce biodiesel
from the algal species [1].The red algae Cyanidium
caldarium is one of the most preferable organism to
produce the oil [2]. Considering the metabolic pathways
of oil production in the algae, the Acetyl-CoA carboxyl
transfersae (ACCase) enzyme plays a major role for oil
production. It catalyzes the production of Malonyl Co-
enzyme a using the substrate Acetyl -CoA and ATP [3].As
the three dimensional structure of the enzyme is not
present in PDB (Protein Data Bank), so an in-silico
approach has been taken to predict the three
dimensional structure by homology modeling method
[4]. By this approach a valid structural model has been
produced with the available template having 46%
similarity. After model generation and validation an
automated version of pocket finding search by CastP
server [5] docking by Hex 5.0 version and structural
motif analysis by EMBOSS tool [6] has been used to
investigate the binding residues responsible for binding
of ATP. Furthermore the in-silico structural analysis
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shows that the residues like 110,113,296 are responsible
for the binding to ATP.
MATERIALS AND METHODS
Sequence retrieval and 3D model building
The sequence for the ACCase enzyme was retrieved from
SWISSPROT database having ID O19903 [7]. Then with
this query sequence a BLAST [8] search was performed
against PDB (Protein Databank) to retrieve the
corresponding template for the ACCase enzyme. The
model was built by homology modeling and for this
MODELLER 9v5 [9] program. The MODELLER
program uses an automated approach to comparative
protein structure modeling by satisfaction of spatial
restraints (Fig.1). Briefly, the core modeling procedure
begins with an alignment of the sequence to be modeled
(target) with related known 3D structures (templates).
This alignment is usually the input to the program. The
output is a 3D model for the target sequence containing
all main chain and side chain non-hydrogen atoms [10].
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Model validation
The MODELLER generated structure was verified for the
missing side chains by SCWRL4 tool [11] further verified
by PROCHECK [12]. The PROCHECK programs
provides the information about the stereo chemical
quality of a given protein structure. The PROCHECK was
used to generate Ramachandrans plot and the quality of
the structure was computed in terms of % of residues in
favorable regions, % of non Proline Glycine residues
etc.The quality of structure was also further accessed by
using ERRAT [13]. ERRAT is a protein structure
verification algorithm that is especially well-suited for
evaluating the progress of crystallographic model
building and refinement. The program works by
analyzing the statistics of non-bonded interactions
between different atom types. A single output plot is
produced that gives the value of the error function vs.
position of a 9-residue sliding window. By comparison
with statistics from highly refined structures, the error
values have been calibrated to give confidence limits.
This is extremely useful in making decisions about
reliability. Then the backbone alignment and RMSD
(root mean square deviation) study was performed by
using PYMOL tool [14]. PyMOL is useful molecular
modeling software allowing the visualization of three
dimensional molecular structures as well as for the
backbone alignment between the model and template.
In-silico conserved motif analysis and binding
pocket analysis of the model
The refined validated model was then subjected to CastP
server to observe the possible pockets in the
protein.CastP server is a automated on line tool that
predicts the possible pockets for along with the residue
positions and surface area.Also to find out the conserved
motif responsible for binding then protein sequence was
searched in EMBOSS tool which scan a protein sequence
with motif from Prosite database.
Docking study and energy minimization
Docking was performed between the validated structure
of ACCase enzyme and the energy minimized ligand by
using HEX 5.0 tool [15]. HEX is an interactive molecular
graphics program for protein-ligand docking, assuming
the ligand is rigid, and it can superpose pairs of
molecules using only knowledge of their 3D shapes. It is
still the only docking and superposition program to use
spherical polar Fourier (SPF) correlations to accelerate
the calculations [16].The binding energy was computed
and the ligand binding pattern was observed. Then a
compared energy minimization was performed in the
model alone and model with the docked ligand by
Chimera tool for 100 steps to observe the stability in
terms of potential energy.
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Raghunath Satpathy et al.
RESULTS AND DISCUSSIONS
Homology modeling of ACCase enzyme
The sequence of 324 amino acid protein AcetylCo-
enzyme carboxylase carboxyl transferase subunit alpha
was obtained from SWISSPROT database. After the
sequence searched in PDB (protein data bank) by
BLAST the template PDB ID 2F9I [17] chain A was
chosen as suitable one as it is having 46 % identity with
the query sequence. Then MODELLER was used to
generate the three dimensional structure (Fig.2) and the
structural features were derived (Table 1). Overall six
models were obtained the suitable model was chosen by
lowest molpdf value.
Refining and Validation of the Model
After getting the model the missing side chains of the
model was checked and managed by SCWRL4 software.
The output PDB file from the software was then verified
by backbone comparison between the template and
refined model. The RMSD of backbone was calculated as
0.210 which is very much reliable.
The modeler generated DOPE score was computed and
analyzed. From the comparative chart (Fig.3) the peak is
showing that there is no defect in the loop regions in the
residues. So in the present case the loop refinement
method is not considered for the model. To verify further
the predicted structures, the coordinates of the predicted
structure was fed into the ERRAT Protein Verification
Server. The overall quality factor was obtained as 78.344
which are very much satisfactory (Fig.4). The stereo
chemical quality of the structure was checked by
PROCHECK tool. The Φ and Ψ distributions of the
Ramachandran plots of non-Glycine, non-Proline
residues are summarized (Fig 5). Altogether 92.3% of the
residues were in favored regions (Table 2) so it can
consider as a good model for further analysis. The
overall G-factor used was computed as- 0.14.
Docking study
Docking of the ATP with the modeled enzyme was
performed using HEX tool. The grid was fixed at 0.6 and
the default FFT (Fast Fourier Transformation) mode was
chosen. The algorithm exhaustively searches the entire
rotational and translational space of the ligand with
respect to the receptors. The ATP binding with the
enzyme was obtained as energy value -228.72.Then the
energy minimization of the only model and the model
complex with ATP was computed by Chimera tool [18]
show the potential energy as -13981 .757 and -15605.920
respectively that indicates the ligand ATP is lodge in its
proper binding site. From the docked structure the
residues that facilitates for binding of the above ligands
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CastP server that also showing the pocket 3 having
residues 110-CB –ILE 110-O-ILE, 113-CB-ARG, 296-
CD2-LEU are responsible for binding (Fig.6). Cross
verified result by EMBOSS tool show that the prosite
motif of the sequence is from residue 110-113(AGRR).
CONCLUSION
Acetyl Co-enzyme carboxyl transferase is the main
enzyme which catalyses the first step of the lipid
synthesis in algae Cyanidium caldarium. The storage
lipids in the algae correspond to the biofuel producing
capability. In the present work a homology based
modeling for the above enzyme has been constructed
using MODELLER software. And after various types of
model validation software the result suggest that the
model is reliable. The stable structure is further
subjected to Docking with ATP the key ligand of the
enzyme action. The docking result shows that almost
similar part of the enzyme is responsible for binding with
both the ligand also the residue. The predicted pocket
result of the enzyme obtained from Cast P server was
found is consistent with the predicted result by EMBOSS
tool for the conserved motif. The work basically relates
to the in-silico analysis of the fatty acid synthetic
pathway leading to accumulation of oils and fats and an
understanding about how the pathway could be utilized
for the commercial production of algal biofuels.
ACKNOWLEDGEMENT
We are thankful to CEO, Director and Dean of
Majhighariani Institute of Technology & Science,
Table - 1
Structural information of the Modeled ACCase enzyme
Structural features
No. of residues
No. of atoms
No. of Hydrogen bonds
No. of helices
No. of strands
No. of turns
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Raghunath Satpathy et al.
Ryagada to provide us the MIRC lab for computing
facility.
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Information
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Table - 2

Ramachandaran plot calculation on 3D model of ACCase enzyme computed with PROCHECK program

% residues in favorable regions 92.3

% residues in additional residue regions 5.2

% residues in generously regions 1.7

% residues in disallowed regions 0.7

% of non Proline and non Glycine residues 100

Figure - 1

Working principle of MODELLER (Sali and Blundell 1993)

Figure - 2

Rasmol view of final 3D structure of the ACCase. Yellow color indicates beta sheets and Red one is the alpha helix.

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Figure - 3

Comparative DOPE value of template and model obtained from MODELLER output

Figure - 4

ERRAT structural quality

Figure - 5

The Ramachandran’s plot for the model protein

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Figure – 6

The docked structure of ATP (Red color) in the pocket residues of the enzyme. (Shown in Blue color)

Citation: Raghunath Satpathy et al., nst Life Sciences and Bioinformatics Vol. 2: 147-152 (2010)

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