Biochemical, Mutational and In Silico Structural Evidence
for a Functional Dimeric Form of the Ornithine
Decarboxylase from Entamoeba histolytica
Preeti1, Satya Tapas1, Pravindra Kumar1, Rentala Madhubala2, Shailly Tomar1*
1Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India, 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Background: Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are
potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by
ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E.
histolytica (EhODC) is reported to exist in a pentameric state.
Methodology and Results: In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-
expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using
circular dichroism spectroscopy. The percentages of a-helix, b-sheets and random coils in EhODC were estimated to be 39%,
25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC
enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two
separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each
active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with
RMSD value ,0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active
site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity.
Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were
mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface
were found to abolish the enzyme activity and destabilize the dimer.
Conclusion: To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form.
These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition
strategies by targeting the dimer disruption.
Citation: Preeti, Tapas S, Kumar P, Madhubala R, Tomar S (2012) Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the
Ornithine Decarboxylase from Entamoeba histolytica. PLoS Negl Trop Dis 6(2): e1559. doi:10.1371/journal.pntd.0001559
Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America
Received September 29, 2011; Accepted January 21, 2012; Published February 28, 2012
Copyright: ? 2012 Preeti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work carried out for this paper was supported by a grant from the Department of Science and Technology (DST), Government of India, New Delhi,
India, to S. Tomar. A Senior Research Fellowship from the Council of Scientific and Industrial Research, India, supported Preeti. A National Doctoral Fellowship
from the All India Council for Technical Education supported S. Tapas. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Amoebiasis is an infectious disease caused by single-celled
parasitic protozoan Entamoeba histolytica. Parasitic amoeba infects
liver and intestine, which may cause mild diarrhea and serious
dysentery with bloody and mucoid stool. If untreated, the parasite
can cause life-threatening hemorrhagic colitis and/or extraintes-
tinal abscesses. E. histolytica is responsible for over 50 million
infections in tropical and temperate regions, and nearly 100,000
deaths worldwide each year [1,2]. The parasite mainly affects
primates and humans, and is transmitted by ingestion of water and
food contaminated with feces containing E. histolytica cysts. First-
line amoebiasis treatment is anti-amoebic therapy that relies on a
very small number of drugs such as metronidazole, emetine,
tinidazole and chloroquine [3–5]. These drugs target different
stages of the life cycle of E. histolytica. Frequent and widespread
usages of these drugs have led to the increase in the minimum
inhibitory concentration (MIC) values and also development of
clinical drug resistance in pathogen. Some of these drugs have
been reported to have significant side effects. For instance,
metronidazole, an effective drug for amoebiasis, has been reported
to be tumorigenic and mutagenic [6–8]. Nitrazoxanide, a broad
spectrum anti-parasitic drug used for amoebiasis treatment, is
found to be associated with many side effects [9,10]. Consequent-
ly, development of alternate strategies and discovery of new anti-
amoebic agents targeting polyamine synthesis is necessary to
combat the disease.
Ornithine decarboxylase (ODC), a Pyridoxal 59-phosphate
(PLP) dependent homodimeric enzyme catalyzes the first rate-
limiting step of polyamines biosynthetic pathway by decarboxyl-
ation of L-ornithine to form putrescine (Figure 1). Polyamines have
an eminent role in various cell growth and differentiation processes
www.plosntds.org1February 2012 | Volume 6 | Issue 2 | e1559
[11,12]. Consequently, ODC being the key enzyme of the
polyamine biosynthetic pathway is a promising therapeutic target
for anti-protozoan therapy. The ODC enzyme has been reported
to be present in various protozoa including Leishmania, Trypanosoma,
Giardia, and Plasmodium and is a validated drug target in
Trypanosoma brucei for treatment of African sleeping sickness [13–
18]. ODC enzyme has a very short half-life due to its ubiquitin-
independent 26S proteasome mediated degradation which is
stimulated by the binding to antizyme . Besides increase in
ODC proteolysis, interaction of antizyme with ODC leads to
catalytic inactivation of the enzyme by disrupting the enzymati-
cally active ODC dimers [19,20]. In addition, the antizyme
binding loop which is accessible in ODC monomer is found to be
buried in the dimers of ODC that ultimately prevents it from
degradation. Thus, dimer formation is not only important for its
catalytic function but also for its protection against antizyme-
Crystal structures of ODC enzyme from T. brucei (PDB ID:
1QU4), human (PDB ID: 2OO0), and mouse (PDB ID: 7ODC)
have revealed that the monomeric subunits interact in head to tail
manner and form two catalytic active sites at the dimer interface
[21–23]. The structure of ODC in complex with substrate and
product analogues including ornithine analog a-difluoromethy-
lornithine (DFMO) have been investigated . DFMO is a
suicide inhibitor of ODC and has been reported to inhibit growth
of various pathogenic protozoan parasites such as Giardia lamblia
, Trichomonas vaginalis , Plasmodium falciparum, and various
Trypanosoma species [13,18]. In E. histolytica, the only enzyme of
polyamine biosynthesis reported to exist is ODC. E. histolytica
ODC (EhODC) has been reported to form homopentamers .
Interestingly, EhODC is insensitive to DFMO and DFMO has no
inhibitory effect on the cell growth of the parasite [25–27].
Therefore, it is necessary to develop an alternate method for
inhibition of EhODC enzyme for targeting the polyamine
biosynthetic pathway to curb the disease.
In the present work, we have re-investigated the oligomeric state
of EhODC using biochemical, mutational and in silico methods.
Previously, it has been reported that the EhODC enzyme exists
only as a homopentamer . However, our studies evidently
demonstrate that EhODC is functionally active in the dimeric
form. In the absence of crystal structure of EhODC, we have
generated 3D model of EhODC homodimer to structurally
characterize the dimer interface containing two active sites and
have performed molecular dynamics simulations to verify the
dimer stability. Our investigation yields that disruption of dimer
disrupts the active site pocket and renders the enzyme inactive. 3D
structure model of EhODC homodimer may be beneficial in
designing structure based anti-amoebiasis peptides or agents that
would disrupt enzyme dimerization. We propose that a compound
having the capability to disrupt the dimer could be a good
candidate for amoebiasis treatment.
Materials and Methods
The E. coli expression vector pET 30a (Novagen) containing
full-length gene of EhODC having N-terminal Histidine tag (66
His) followed by enterokinase cleavage site was used for over-
expression of the enzyme . Oligonucleotides for site directed
mutagenesis were ordered from Imperial Life Sciences (India).
Restriction endonuclease DpnI and Phusion DNA polymerase
were acquired from New England BioLab Inc. For protein
purification, 5 ml HisTrap HP and HiLoad 16/60 Superdex 200
gel filtration columns were obtained from GE Healthcare.
Imidazole (low absorbance at 280) was obtained from Acros.
A¨KTA Prime plus system from GE Healthcare was used for
protein purification. Putrescine, 4-aminoantipyrine, diamine
oxidase (DAO), horseradish peroxidase, L and D-ornithine were
procured from Sigma Aldrich. Amicon ultra protein concentrators
were purchased from Millipore. All other chemicals were of
analytical grade and obtained from commercial sources.
Over-expression and purification of recombinant EhODC
The expression and purification of EhODC enzyme was done
by following the published procedure with minor modifications
Figure 1. The enzymatic reaction catalyzed by ornithine decarboxylase. The pyridoxal phosphate (PLP)-dependent ODC enzyme catalyzes
decarboxylation of ornithine and produces putrescine.
E. histolytica genome sequence divulged the existence of
ornithine decarboxylase enzyme that performs the first-
rate limiting catalytic step of polyamine biosynthetic
pathway. ODC enzyme is a potent therapeutic target in
many eukaryotic disease causing pathogens. DFMO, a
potent substrate analogue inhibitor, is widely used for the
treatment of various diseases including Trypanosoma
brucei infections. However, DFMO does not inhibit E.
histolytica ODC. As ODC is a validated drug target for
protozoan disease, an alternate strategy to inhibit the
EhODC enzyme may be developed. In our study, we have
evidently proved that the purified recombinant EhODC is
functional as an active homodimer. Molecular modeling
and simulation studies indicate that two independent
active sites are present at the dimer interface. Our
mutational studies indicate that the enzyme activity can
be abolished by targeting the dimer interface and this in
turn suggests the alternative inhibitory mechanism for the
enzyme. Our investigation yields that disruption of dimer
disrupts the active site pocket and renders the enzyme
inactive. As EhODC crystal structure is unavailable, the 3D
structure model of EhODC homodimer may assist in
designing structure based anti-amoebiasis peptides or
agents that disrupt the active site by destabilizing the
Evidence for Functional Dimeric Form of EhODC
www.plosntds.org2February 2012 | Volume 6 | Issue 2 | e1559
given below . The plasmid pET30a having the full-length
EhODC gene insert (pET30a-EhODC) was transformed into
freshly prepared E. coli BL21 (DE3) competent cells and plated on
Luria-Bertani (LB) agar plate containing kanamycin (50 mg/ml).
Plates were incubated overnight at 37uC and colonies were
obtained. Single colony was picked and cells were seeded in 5 ml
LB broth containing 50 mg/ml of kanamycin and culture was
grown overnight at 37uC with agitation. Overnight culture was
used for inoculation of 1 L LB broth. Expression was induced with
1 mM isopropyl b-D-thiogalactoside (IPTG) when optical density
(A600) reached 0.6. After induction, culture was moved to 18uC
and was grown for ,14 h. Cells were harvested by centrifugation
at 5,000 rpm at 4uC for 10 min and cell pellets were stored at
280uC until further processing. Expression and solubility of the
protein was confirmed by analysis of lysed cell supernatant and
pellet on 12% sodium dodecyl sulfate-polyacrylamide gel electro-
The histidine-tagged EhODC was purified using a two step
procedure that employed metal ion affinity chromatography
followed by gel filtration chromatography. All purification steps
were performed at low temperature (4uC–6uC). Briefly, frozen cell
pellets from a 1 L culture were thawed on ice and re-suspended in
buffer A [50 mM Tris-HCl (pH 7.5), 40 mM imidazole, 250 mM
NaCl and 5% glycerol (v/v)] containing lysozyme (0.7 mg/ml) and
0.2 mM phenylmethanesulfonyl fluoride (PMSF). Cells were
disrupted by sonication on ice with a pulse of 20 s on and 1 min
off for 10 times. The obtained cell lysate was clarified by
centrifugation at 18,000 g for 45 min at 6uC and supernatant
was applied on HisTrap HP column (5 ml) pre-equilibrated with
buffer A. Unbound proteins were removed by washing the column
with ,40 ml of buffer A. Bound protein fractions were eluted
using a linear gradient of 40 mM to 1 M imidazole of 60 ml at a
flow rate of 1 ml/min. Eluted fractions were examined on 12%
SDS-PAGE and fractions containing pure protein were pooled
together. To remove the N-terminal His-tag, enterokinase was
added to pure protein (,0.02 units/mg protein) and incubated for
,12 h at 4uC and simultaneously dialyzed against buffer A
without imidazole. To remove uncleaved tagged protein and the
cleaved His tags, the sample was reloaded onto HisTrap HP
column and the flow-through containing untagged EhODC was
collected and concentrated using a 10 kDa cutoff Amicon Ultra-15
concentrator (Millipore, Bedford, Massachusetts, USA). For
removal of enterokinase, the concentrated sample was loaded
onto HiLoad 16/60 prep grade Superdex 200 size-exclusion
chromatography column pre-equilibrated with buffer B [50 mM
Tris-HCl (pH 7.5), 250 mM NaCl, 0.2 mM dithiothreitol (DTT)
and 5% glycerol (v/v)]. Fractions of the major peak containing
pure protein were pooled and concentrated. Homogeneity of the
concentrated enzyme preparation was analyzed by 12% SDS-
PAGE. The yield and concentration of purified EhODC was
measured using the Bio-Rad protein-assay kit with bovine serum
albumin (BSA) as a standard. EhODC mutant proteins were
expressed and purified using the same protocol.
EhODC enzyme assay
Ornithine decarboxylation activity of EhODC was spectropho-
tometrically determined by the method developed by Badolo et al.
. This method is based on the reaction between DAO and
putrescine, the product of the ODC-catalyzed reaction. For
EhODC enzyme assay, the purified protein was buffer exchanged
with 20 mM sodium phosphate buffer (pH 7.5) and concentrated
to final concentration of 0.3 mg/ml. The reaction mixture of
180 ml containing 20 mM sodium phosphate buffer (pH 7.5),
0.1 mM EDTA, 0.1 mM PLP, 0.2 mM DTT, and 1 mM of
L-ornithine was prepared to which 20 ml of protein solution was
added to make up the final volume of 200 ml. The reaction
mixture was incubated at 37uC for 5 h. Further, 100 ml of the
above EhODC reaction mixture was added to 900 ml of diamine
oxidase (DAO) reaction mixture composed of 50 mM Tris-HCl
(pH 9.8) containing 100 mg/ml phenol, 100 mg/ml 4-aminoanti-
pyrine (4-AAP), 0.02 U of DAO, and 7 U of horseradish
peroxidase (HRP). The reaction was incubated at 25uC for
60 min and then terminated by heating the solution at 90uC for
4 min. The concentration of putrescine formed by ornithine
decarboxylation catalysis was determined by measuring the
absorbance at 492 nm for the colored complex formed as a result
of the reaction of H2O2with 4-AAP and phenol catalyzed by
HRP. For negative controls, purified protein or substrate L-
ornithine were substituted with buffer in the ODC enzyme
reaction mixtures. Effect of stereoisomer of substrate was observed
by incubation of L and D-ornithine at 37uC.
To obtain preliminary information on the oligomeric associa-
tion of EhODC, glutaraldehyde crosslinking experiment was
performed using the method described by Fadouloglou et al.
. Purified protein solution was exchanged with 20 mM sodium
phosphate buffer (pH 7.5) for cross-linking studies. Experiment
was carried out using 24 well crystallization plate (Hampton
research) and a siliconized coverslip in a manner similar to a
hanging drop crystallization method. For cross-linking EhODC,
40 ml of 12.5% glutaraldehyde solution (v/v) acidified with 1 ml 5
N HCl was added in the well of crystallization plate. Then, 15 ml
of protein solution (1 mg/ml) was loaded onto the coverslip, which
was inverted on the reservoir well and sealed with vacuum grease
(Hampton Research). The entire setup was incubated at 37uC for
10 min and then the sample was mixed with an equal volume of
2X SDS-PAGE loading buffer and boiled for 4 min on a dry bath.
Cross-linked oligomers were analyzed on 12% SDS-PAGE
followed by Coomassie Blue R-250 staining.
Molecular mass and oligomeric state determination
The molecular mass of recombinant EhODC was determined
by running purified protein on 12% SDS-PAGE with standard
molecular weight protein marker (Bio-Rad). To analyze the
oligomerization state, 500 ml of purified and concentrated
(,10 mg/ml) protein was applied onto a HiLoad 16/60 Superdex
200 gel filtration column pre-equilibrated with buffer B using
500 ml sample loop at a flow rate of 0.5 ml/min on A¨KTA purifier
chromatographic system (GE Healthcare) and protein elution
profile was monitored by measuring the absorbance at 280 nm.
The size-exclusion column was calibrated with blue dextran
(2000 kDa), and Gel Filtration HMW Calibration kit containing
ferritin (440 kDa), aldolase (158 kDa), Conalbumin (75 kDa) and
ovalbumin (43 kDa) (GE Healthcare) for determination of the void
volume, construction of the standard curve and estimation of the
molecular weight of purified protein.
The oligomerization state of EhODC was also analyzed by
matrix-assisted laser desorption/ionization time of flight mass
spectrometry (MALDI/TOF MS). The purified protein sample
was dialyzed against 50 mM Tris buffer (pH 7.5) containing low
concentration of NaCl (25 mM) and 0.2 mM DTT to avoid any
instrumental interference and was concentrated to ,2 mg/ml
using 10 kDa cutoff Amicon ultra 15 (Millipore). The MALDI/
TOF MS analysis was carried out at Proteomics Facility, TCGA
(New Delhi, India) using Ultraflex mass spectrometer (Bruker
Daltonics, Germany). The protein ionization spectra were
Evidence for Functional Dimeric Form of EhODC
www.plosntds.org3 February 2012 | Volume 6 | Issue 2 | e1559
analyzed on FLEX-PC2 mass spectrometer and data was acquired
across the range of about 0 to 250 amu.
Effect of Urea and NaCl on EhODC oligomerization
To study the effect of urea and NaCl on oligomeric state of
protein, purified and concentrated EhODC was pre-incubated
with variable concentration (2 M or 4 M) of above chemical
agents separately at 4uC for 4 h. The protein was further loaded
onto Hi-load 16/60 superdex 200 gel filtration column equili-
brated with Buffer B containing the same concentration of urea or
NaCl and elution profiles were analyzed.
Far-UV Circular Dichroism spectrum
For estimation of secondary structure elements, purified
EhODC was subjected to circular dichroism (CD) analysis using
Chirascan Circular Dichroism Spectrometer (Applied Photophy-
sics Ltd., Surrey KT22 7PB, United Kingdom). CD spectra were
collected using a 1 mm quartz cell under constant nitrogen purge
between 190 to 260 nm in 0.5 nm wavelength steps and an
average time of 3.0 s at 25uC. The protein solution was buffer
exchanged with 20 mM potassium phosphate buffer (pH 7.5) at
4uC. Protein samples at concentration 0.35 mg/ml were analyzed
and three scans were collected, averaged and the baseline
corresponding to the above buffer was subtracted to obtain the
final values. The obtained data were analyzed using the software
K2d (http://www.embl.de/,andrade/k2d.html) .
Site directed mutagenesis
The pET30a-EhODC plasmid containing EhODC gene was
mutated using the QuikChange XL mutagenesis kit by following
the instructions of manufacturer (Stratagene, La Jolla, CA).
Mutations were introduced into the synthetic mutagenic oligonu-
clotide primers and were used for construction of mutant plasmids.
Mutations and respective mutagenic primers are listed in table
(Table 1). The pET30a-EhODC plasmid was used as a template in
the primer extension reaction for constructing the mutants. The
reaction mixture used for PCR amplification contained 10 ml of
5X HF phusion buffer supplied with the enzyme, 300 mM of
dNTP mix, 6.25 pmol of each primer, 10 ng of template DNA,
2.5 U of phusion polymerase, and water was added to make up the
final volume of 50 ml. PCR reaction was performed by subjecting
the samples to 20 cycles of 30 s denaturation at 95uC, 1 min at
annealing temperature as given in Table 1, and 6 min 50 s
elongation at 72uC, and finally reaction was completed by doing
extension for 15 min at 72uC. PCR products were analyzed on 1%
agarose gel electrophoresis. The parent methylated template
plasmids were digested with DpnI restriction enzyme at 37uC for
1 h. Digested product was directly used to transform XL-1 Blue
competent cells. Transformed cells were plated on LB agar plate
containing 50 mg/ml of kanamycin and plates were incubated at
37uC for ,16 h. The presence of the mutations in the constructed
plasmids were confirmed by DNA sequencing using T7 promoter
or terminator universal primers at genomic and proteomic facility
of TCGA (New Delhi, India).
Phylogenetic and sequence analysis
The ODC sequence of E. histolytica was retrieved from NCBI
database. Blast and PSI-blast search were performed using
AAX35675.1 as query against the non redundant protein
sequence database to identify and analyze orthologous sequences.
These homologous sequences were retrieved from the NCBI
database and multiple sequence alignment was generated using
ClustalW and compared for phylogenetic analysis .
Three-dimensional (3D) homology model of EhODC homodi-
mer was generated by comparative modeling using MODELLER
9v8 . To obtain an effectual model, five sequential steps were
performed: template selection from Protein Data Bank (PDB),
sequence-template alignment, model building, refinement of the
obtained model and validation. Template search was done using
NCBI BLAST search tool for PDB database . BLASTP
algorithm was run with BLOSUM62 as a scoring matrix. Crystal
structure of human ODC (PDB ID: 2OO0) which has 34%
sequence identity with EhODC was selected as a template for
structure modeling . The graphically enhanced alignment with
secondary structures were obtained using ESPript 2.2 server .
MULTALIN server was used to align the query sequence with
the template sequence . Some manual corrections were done
in the alignment file for missing residues in the template sequence.
The cofactor, PLP was incorporated into the modeled structure of
EhODC from the template structure and five preliminary models
were generated using MODELLER 9v8. All models were selected
on the basis of lowest DOPE scores and assessed sterio-chemically
by PROCHECK . Energy minimizations of the best chosen
models were performed using Swiss-PDB Viewer4.01 (http://
www.expasy.org/spdbv/). Loop refinement module of the MOD-
ELLER was used for the refinement of the disorganized residues in
loops and refinement process was considered for structure
validation. Each refined model was verified using ERRAT plot
which gives the measure of structural errors in each model at
residue level in the protein (http://nihserver.mbi.ucla.edu/
Table 1. Sequence of mutagenic primers and annealing temperature used for PCR amplification of mutant plasmids.
MutantsNucleotide sequenceAnnealing Tm
Lys57Ala (S) CTTGCTTTGCTGTTGCATGTAATCCTGAACCTCA
Lys57Ala (An) TGAGGTTCAGGATTACATGCAACAGCAAAGCAAG
Gly361 Tyr(S) GGTTATTATTTCCCAATATGTATGCTTATACAATTTC
Lys157Ala (S) ATGTATTTGGAGAGGCATTTGGACTTCATGATGA
Lys157Ala (An) TCATCATGAAGTCCAAATGCCTCTCCAAATACAT
Mutated nucleotides are underlined. S: sense and An: antisense.
Evidence for Functional Dimeric Form of EhODC
www.plosntds.org4February 2012 | Volume 6 | Issue 2 | e1559
SAVES/). The refined model was further validated by ProSA
energy plot and VERIFY-3D of the SAVES server [36,37]. All the
graphical visualization and image production were performed
using PyMOL .
Molecular dynamics simulation
Molecular dynamics (MD) simulation of dimeric model of
EhODC was performed using GROMACS (v 4.5.4) package .
GROMOS96 43a1 force field and 47324 SPC water molecules for
solvation of protein were used for simulation. The molecule was
solvated in a cubic box at a distance of 1.0 nm between the
proteins and the box edge. Electrostatic interactions were
calculated using the Particle-mesh Ewald method. Van der Waal
and coulomb interactions were truncated at 1 nm. Molecule was
neutralized by adding 24 Na+counter ions to the surface and was
allowed to undergo 1000 energy minimization steps. All bond
lengths including hydrogen atoms were constrained by the LINCS
algorithm. To maintain the system at isothermal and isobaric
conditions of 300 K and 1 bar, a V- rescale and Parrinello-
Rahman barostat coupling was applied for 100 ps. Following to
the equilibration, MD simulation was initiated for 1 ns and then
extended to 8 ns, with all trajectories sampled at every 1.0 ps.
Results and Discussion
Sequence analysis and phylogeny
The completion of genome sequence project of E. histolytica
headed by the Institute of Genome Research (TIGR, Rockville,
USA.) opened up the possibilities of new therapeutic targets as well
as detailed mechanisms of various biosynthetic pathways . The
polyamine biosynthesis in E. histolytica is an essential pathway
required for the existence of the pathogen [11,12]. In present
study, the sequence of EhODC, the first and rate-limiting enzyme
of polyamine biosynthetic pathway, has been retrieved from NCBI
database with accession number AAX35675. The protein consists
of 413 amino acids with predicted molecular weight of 46.43 kDa.
In E. histolytica, the gene encoding ODC is of 1242 bp, thus it
implies that there is no intron present in the gene. The enzyme has
been previously characterized by Jhingran et al. . The amino
acid sequence alignment of EhODC with representative ODCs
from different sources revealed that the active site residues along
with dimer interface residues responsible for dimerization are
highly conserved (Figure 2). EhODC showed overall 36 to 39%
identity with plants, 15 to 25% with bacteria, 35 to 38% with fungi
and 32 to 38% with animals. Interestingly, E. histolytica, being a
protozoan was expected to show high sequence identity, but
Figure 2. Multiple sequence alignment of EhODC (AAX35675) with other ODC sequences. The conserved residues are highlighted with
black background color. The secondary structure elements and numbering of amino acid sequence of human ODC are presented above the aligned
sequences. The signatory motifs PxxAVKC(N) (PLP binding motif) and WGPTCDGL(I)D (substrate binding motif) are highlighted in boxes where ‘‘x’’
signifies any amino acid and amino acids in brackets depict the option at a given position. Underlined sequence denotes the amino acids showing
similarity with (1) Antizyme binding region (2) PEST like region. The circles under the amino acid indicate the residues interacting with cofactor PLP
where as triangles denote the substrate L-ornithine binding residues in the active site pocket. The residues denoted with cross mark are involved in
formation of salt bridges in between two monomers. The residues indicated with stars are present at the interface and form a stack of aromatic rings.
Residue important for dimer formation and present away from the interface is denoted with a square. The motif A represents the interface residues of
two monomers present very closer to each other. Alignments are obtained using ESPript.
Evidence for Functional Dimeric Form of EhODC
www.plosntds.org5 February 2012 | Volume 6 | Issue 2 | e1559
surprisingly it shows same range of identity with other protozoa
including T. brucei, Dictyostelium dasciculates and P. falciparum, etc. i.e.
32 to 35%. From phylogenetic tree, the ODC from plants, fungi,
and bacteria make different clusters on the basis of sequence
homology where as the protozoan ODCs do not cluster together,
instead are distributed throughout showing resemblance with
bacteria, fungi and plants (Figure 3). However, EhODC shows
maximum homology with plant ODCs and the evolutionary origin
of EhODC or protozoan ODCs on the basis of phylogenetic
analysis is not conclusive. Nevertheless, sequence analysis shows
conservation of dimer interface residues which specify the
possibility of EhODC enzyme dimerization similar to other ODCs.
Further sequence analysis revealed that the substrate binding
motif having a consensus sequence WGPTCDGL(I)D is highly
conserved in human, mouse and T. brucei and Cys plays a critical
role in catalysis. However, in EhODC, though Cys is conserved,
but the sequence exists as 330 YGPSCNGSD 338 (Figure 2).
The regulation of ODC activity is partially modulated by
antizyme-induced, ubiquitin-independent degradation by the 26S
proteasome, mainly found in mammals [20,41–43]. Antizyme
binds to the inactive ODC monomer forming a hetero-dimer
complex which promotes proteolysis degradation [20,44]. In
human ODC, the antizyme binding locus consists of 30 residues at
N-terminal ranging from 115Lys to 144Arg residues. The same
locus is also highly conserved in mouse. However, this locus in
EhODC which corresponds to 105Tyr to 132Lys having 23%
identity is not conserved. In this locus, three residues 121Lys,
141Lys and 144Arg (in human ODC) are highly conserved and
responsible for antizyme binding . However, in EhODC,
121Lys and 144Arg are substituted by 109Ile and 132Lys
Figure 3. Phylogeny of ornithine decarboxylase from various sources. The amino acid sequences of ODC were taken from plants R.
communis (XP_002510610.1), N. glutinosa (AAG45222.1), C. annum (AAL83709.1), Z. mays (AAM92262.1), D. stramonium (P50134.1); animals X. laevis
(NP_001079692.1), R. norvegicus (NP_036747.1), M. musculus (P00860.2), H. sapiens (P11926.2); fungi A. oryzae (XP_001825149.2) M. circinelloides
(CAB61758.1), E. festucae (ABM55741.1), P. brasiliensis (AAF34583.1), S. cerevisiae (EDN60096.1) F. solani (ABC47117.1), C. albicans (AAC49877.1);
protozoa P. bursaria (NP_048554.1), T. brucei (P07805.2), L. donovani (P27116.1), E. histolytica (AAX35675) and bacteria V. vulnificus (YP_004188159.1),
A. caulinodans (YP_001523249.1), P. syringae (AAO58018.1), E. amylovora (YP_003539917.1), S. scabiei (YP_003491041.1), Azospirillum (BAI72082.1),
E. coli (BAE77028.1), Lactobacillus (P43099.2). Different clusters representing a particular group are highlighted in boxes where as the representatives
of protozoa ODC are highlighted by arrow marks.
Evidence for Functional Dimeric Form of EhODC
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respectively. Thus, it may be possible that these differences in
sequence makes EhODC insensitive or poorly sensitive to
antizyme binding as antizyme dependent ODC degradation has
not been reported in E. histolytica till date.
Addition to this, in mouse ODC two basal degradation elements
(376 to 424 and 422 to 461) at C-terminal are reported which are
rich in proline (P), glutamic acid (E), serine (S), and therionine (T)
called PEST sequence . In this region, C441 (in both mouse
and human ODC) is identified to be a critical residue that
promotes polyamine-dependent proteolysis [20,45]. Similar pat-
tern of sequence arrangement is also observed in EhODC where it
ranges from 395 to 413, and conserved Cys400 corresponds to
Cys441 in mouse ODC.
EhODC purification and enzyme activity
The recombinant EhODC protein was purified to homogeneity
using two step procedure consisting Ni2+affinity chromatography
and size exclusion chromatography. The crude containing over-
expressed EhODC from E. coli having N-terminal His-tag was
loaded onto HisTrap Ni2+column and eluted using a linear
gradient of imidazole. The N-terminal His-tag from eluted protein
sample was removed using enterokinase and sample was re-loaded
onto HisTrap Ni2+column. Then, the flow-through containing
EhODC without His-tag was collected, concentrated and loaded
onto HiLoad 16/60 superdex 200 gel-filtration column for further
purification. Homogeneity of pure protein sample was estimated
on 12% SDS-PAGE, which exhibited a single band of ,46 kDa
corresponding to the molecular weight of EhODC protein
(Figure 4). The yield of the purified protein was estimated to be
,3.0 mg/L of culture and protein was concentrated to ,6 mg/
The enzymatic activity of purified protein was demonstrated
using the simple and rapid colorimetric ODC activity assay .
The decarboxylation activity of purified enzyme was assayed in
200 ml reaction containing 20 mM sodium phosphate buffer
(pH 7.5), 0.1 mM EDTA, 0.1 mM PLP and 1 mM of L-ornithine.
The reaction was assayed in terms of the formation of product,
putrescine by its oxidation using DAO enzyme which releases
H2O2that forms a colored complex as described in materials and
methods. His-tagged and untagged protein showed no difference
in the enzymatic activity. Furthermore, the purified EhODC
actively catalyzed the conversion of L-ornithine to putrescine,
while it showed no activity when D-ornithine was used as a
substrate in enzyme reaction. This reveals that EhODC enzyme is
stereospecific in binding to L-ornithine substrate suggesting that
substrate based stereospecific inhibitors may be designed for
Secondary structure analysis of EhODC
An effort was made to elucidate the secondary structure of
EhODC by using Far-UV circular dichroism (CD). CD spectrum
analysis of EhODC exhibits two negative peaks at 211 and 219 nm
and a positive peak in the range of 192-203 nm, as expected for a
protein with a/b content, indicating that purified protein has a
well defined structure (Figure 5). The deconvolution of CD data
with K2d program indicates a secondary structural content of 39%
a-helix, 25% b-sheet, and 36% random coil (http://www.embl.
de/,andrade/k2d.html) . For comparative secondary struc-
ture analysis, the server SOPMA was used for the prediction of
secondary structural elements in EhODC sequence . K2d
results were found to be in agreement with the result of SOPMA
showing 33% a-helix and 25% b-sheet content (Figure 5). These
estimations are in accordance with the available crystal structures
of ODCs and also with the molecular model for EhODC, which
was generated by homology modeling in the present study. These
results reveal that EhODC contains an a/b tertiary structure and
has the overall folding pattern similar to the other ODCs from
mammals, plants and protozoa.
Characterization of oligomeric state of wild type EhODC
ODC purified from E. histolytica has previously been reported to
exist in a pentameric state . Three dimensional crystal
structure studies of ODCs from different sources have shown that
the enzyme exists as a homodimer and association of monomeric
subunits directs the formation of two equivalent catalytic pockets
Figure 4. Purification and molecular mass determination of
EhODC. (A) Affinity purification of EhODC showing purified protein in
12% SDS-PAGE. Lane 1: Molecular weight marker; Lane 2: Purified
EhODC-His tagged protein; Lane 3: Purified His tag cleaved protein with
molecular weight ,46 kDa. (B) Size-exclusion chromatography profile
of EhODC and 12% SDS-PAGE (insert) analysis of major peak fractions.
(C) The elution profile of standard molecular weight markers from size
exclusion chromatography through HiLoad 16/60 Superdex 200
column. The column void volume (Vo) and molecular weight (kDa) of
standard proteins are indicated.
Evidence for Functional Dimeric Form of EhODC
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at the dimer interface. Structural analysis revealed that each active
site at the dimer interface is assembled by amino acid residues
contributed from each monomer subunit, which has also been
confirmed by mutational studies [21–23]. Therefore, we were
interested in characterizing the functional oligomeric form of
EhODC. To accomplish this, we purified recombinant EhODC
enzyme and first confirmed that the purified protein is
Cross-linking agent, glutaraldehyde is used for obtaining crude
information about the quaternary structure of proteins .
Previously, the crosslinking experiment has been performed to
reveal the dimeric form of mouse ODC [47,48]. Therefore,
EhODC was cross-linked using glutaraldehyde in a closed setup
similar to protein hanging drop crystallization method. After
incubation for 10 min, the protein sample was analyzed using
SDS-PAGE. The cross-linked sample showed two bands of
,90 kDa and ,46 kDa corresponding to the molecular weight
of EhODC dimer and monomer (Figure 6) indicating the
possibility of EhODC dimerization.
To further analyze EhODC oligomerization, the molecular
weight of purified protein was estimated by applying the sample
onto a HiLoad 16/60 prep grade Superdex 200 gel-filtration
column using A¨KTA purifier. Purified protein showed a major
peak with the elution volume 71.3 ml (Figure 4). Using a standard
curve based on molecular weight markers, the molecular weight of
major elution peak was calculated and was estimated to be
approximately ,90 kDa, which corresponds to the molecular
weight of EhODC dimer (Figure 4). This suggests that EhODC
exists in the dimeric form. Furthermore, MALDI/TOF MS
analysis of the purified protein was carried out to verify and
confirm the dimerization of protein. MS data showed two narrow
peakshavingaverage intensity of44558.430 m/zand
90667.295 m/z and these correspond to the monomeric and
dimeric state of the protein respectively (Figure 6). Thus, it was
established that EhODC enzyme exists in dimeric state.
Figure 5. Circular Dichroism spectroscopy of EhODC. A Far-UV CD spectrum of 0.35 mg/ml EhODC. Data was analyzed using online K2d server
for determining the secondary structure contents. Inserted table shows the comparative secondary structure content obtained by CD data analysis
and SOPMA server.
Figure 6. Oligomeric state determination. MALDI-TOF MS analysis
of EhODC showing two peaks corresponding to ,44558.430 Da and
,90667.295 Da. The insert shows 12% SDS-PAGE analysis of glutaral-
dehyde crosslinked EhODC. Lane 1: Molecular weight markers; Lane 2–3:
Protein treated with glutaraldehyde and the two bands correspond to
dimer (,90 kDa) and monomer (,46 kDa). Arrow points to the
crosslinked dimer of EhODC; Lane 4: Purified protein not treated with
Evidence for Functional Dimeric Form of EhODC
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The study of effect of chaotropic agents on oligomeric state is
critical to evaluate the stability of quaternary structure of
proteins. The behaviour of ODC in presence of such agents
differs from species to species and dissociation of oligomeric
state is dependent on the concentration of chaotropic agents
[49,50]. In T. brucei, ODC dissociates into monomers in
presence of high concentration of salt and urea . This
provoked us to examine the effect of different concentrations of
NaCl and urea on oligomeric state of EhODC. Incubation of
protein sample with 2 M and 4 M of NaCl resulted in partial
dissociation of dimeric enzyme to monomeric state (Figure 7).
Two peaks were observed in gel filtration chromatogram: one at
71 ml elution volume followed by a smaller peak at 81 ml
elution volume which correspond to the molecular mass of the
dimeric and monomeric forms of EhODC respectively (Figure 7).
With increased concentration of NaCl from 2 M to 4 M, the
small peak corresponding to monomer becomes more distinct
demonstrating that higher concentration of NaCl partially
disrupts the dimerization. This also suggests the role of inter-
molecular salt-bridges and weak polar interactions in EhODC
dimerization. Similar results were observed when the protein
was treated with 2 M and 4 M urea (Figure 7). Destabilization
of EhODC dimers in higher urea concentration points to the
presence of inter-molecular hydrophobic interactions at the
Generation and stability of 3D molecular model of
The molecular structure and subunit interactions in EhODC
were investigated by constructing a dimeric model of the enzyme
using homology modeling approach. The sequence homology
search for EhODC gave the hits of 29 sequences against PDB
database. The crystal structure of human ODC was the first hit
with 34% sequence identity (PDB ID: 2OO0) followed by TbODC
(33%, PDB ID 1QU4). For comparative homology modeling, it
could be significant to select a template for ODC from protozoan
source i.e. TbODC. However, too much variations in the
sequences of ODC within protozoa (Figure 2) and higher sequence
identity of EhODC with plant and mammalian ODC, give an
indication of caution required in the interpretation of template
selection. Here, we have selected human ODC as template for a
reliable model generation considering two major facts: firstly, the
N-terminal loop region consisting of approximately eight amino
acids is missing in all crystal structures of ODC except human
ODC. Secondly, multiple sequence alignment analysis showed a
PEST like sequence in the C-terminal region of EhODC sequence
that has maximum similarity with human ODC (Figure 2). The
model for EhODC along with its cofactor PLP was generated from
PDB 2OO0 as a template using Modeller 9v8 and model with
lowest DOPE score was considered for further loop refinement
using Modeller loop refinement tool. The model was subjected to
Figure 7. Effect of chaotropic agents on oligomeric property of EhODC. (A) & (B) Gel-filtration chromatogram showing the elution profile of
EhODC protein treated with 2 M and 4 M NaCl respectively; (C) & (D) Gel filtration chromatogram showing the profile of protein treated with 2 M and
4 M urea respectively.
Evidence for Functional Dimeric Form of EhODC
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energy minimization where PROCHECK, ERRAT plot and
ProSA energy plot were used for validation and quality assessment
of the model. The root-mean-square deviation (RMSD) of Ca
atoms between the modeled EhODC dimeric structure and the
template structure was 0.744A˚. Ramachandran plot of the model
generated by PROCHECK showed 90.3% residues in the core
region, 7.8% in allowed region, 0.6% in generously allowed region
and 0.3% in disallowed region. The generated models have been
submitted to Protein Model database (PMDB) with PMDB id:
PM0077698 (monomer) and PM0077699 (dimer).
The molecular model of EhODC dimer that was generated
using the crystal structure of human ODC dimer as a template was
MD simulated for 8 ns in equilibration with water molecules.
Evaluation of the dimer stability was made by monitoring the root-
mean-square deviations (RMSD) of the Ca of the dimer which was
computed against the starting structure. Analysis of MD trajectory
of EhODC homodimer revealed that RMSD value increases to
0.327 nm in about 1.2 ns and this plateau value is stable till the
end of the simulation indicating a stable conformation of the dimer
(data not shown).
Structure analysis of EhODC monomeric subunit
Structure of EhODC monomer subunit is comprised of two
major domains i.e. b/a-barrel and b-sheet domain which is a
distinct characteristic of ODC structure (Figure 8). In human
ODC, N-terminal starts with a b-strand while in EhODC, it starts
with a-helix. The N-terminal emerges from b-sheet domain and
enters the barrel through a coil connecting both the domains. The
barrel contains eight parallel strands each followed by a helix in
the order a2b2, g1a3b3, a4g2b4, a5b5, a6b6 a7b7, a8b8 and
a9g3b9. One important feature observed in EhODC is the
presence of turns in a pattern at the N-terminal barrel secondary
structures. Such pattern has been observed in ODC like antizyme
inhibitor proteins that have structures similar to ODC, but do not
possess decarboxylation activity . The sheet domain is
subdivided into two clusters of sheets S1 and S2 as observed in
all ODC structures. These sheets S1 and S2 remain perpendicular
to each other having four helices with one turn (a1, a10, a11, a12
and g4) around it. Sheet S1 includes three parallel b-strands
(Qb11, qb12and qb13) which extends into S2 containing four
parallel b-strands (Qb10, qb14, qb15and qb1) (Figure 8).
Structure analysis of dimeric EhODC
In the dimeric structure of enzyme, two active site pockets rest
at the dimer interface involving the interactions of residues from
both the subunits. b/a-barrel domain is the main site for cofactor
PLP binding where as residues from the sheet domain of other
subunit interacts with the substrate L-ornithine to form the
complete catalytic pocket for enzymatic activity. The subunits
associate in a head to tail manner (Figure 9). The dimeric structure
Figure 8. 3D structure of EhODC monomer. (A) Cartoon diagram of EhODC model generated using Modeller 9v8. (B) Topological arrangement of
secondary structures in EhODC monomer. Monomer of EhODC consists of two domains, b/a-barrel shown in purple and sheet domain having sheet
S1 in green, sheet S2 in blue and helices and turns in orange. The helices are presented by circles, strands are represented by triangles and the loops
connecting these structures are represented as connecting lines.
Evidence for Functional Dimeric Form of EhODC
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Figure 9. Schematic representation of dimer interface and active site of EhODC. (A) Subunits of the dimer are arranged in head to tail
manner where subunit A and B are shown in yellow and green colors respectively. (B) The residues critically important for dimer formation are
presented in sticks and overall dimeric structure is presented in cartoon. Residues from opposite monomer are marked by apostrophe (’) sign. (C)
Surface view of monomeric chains highlighting the residues at the dimer interface in different colors. The monomers have been separated and
rotated to 90u giving clear view of interface residues. Red and blue color indicates residues involved in salt bridge formation and orange color depicts
hydrophobic interactions. (D) Closer view of residues at the interface forming salt bridge. (E) Aromatic residues at the interface arranged as a stack of
ring structures forming amino acids zipper. (F) Residues at the active site interacting with cofactor PLP from each monomer are presented in sticks.
Residues from subunit A and B are shown in yellow and green colors respectively.
Figure 10. Enzyme activity of wild type EhODC and its mutants. Enzymatic activity of EhODC mutants relative to the activity of the wild-type
enzyme. Cys334Ala, Lys57Ala Gly361Tyr and Lys157Ala are inactive. Cys334Ala and Lys57Ala mutants were mixed in 1:1 ratio and the mixture shows
recovery of approximately 29% of the wild-type enzyme activity. The plot represents the average of three measurements.
Evidence for Functional Dimeric Form of EhODC
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is stabilized by various polar interactions present between the two
subunits at the dimer interface as shown in figure 9. However, four
major salt bridges K157-D3389 and D122-R2779, D338-K1579
and R277-D1229 are observed and these have been reported to
play a vital role in the dimer formation of human, mouse, and T.
brucei ODCs . These interface residues are partially hydro-
philic and are highly conserved in human, mouse and EhODC.
Furthermore, the most prominent feature observed near C-
terminal domain is presence of a stack of aromatic rings i.e.
F3719/H2969/F305 and F3059/H296/F371 which is anticipated
to function as an amino acid zipper. Distal amino acid residues of
the zipper participate in active site pocket formation. Further, the
structural analysis revealed that the close packing of dimers shields
the putative N-terminal antizyme binding loop (residues 105Tyr-
132Lys) as well as the C-terminal PEST like sequence because
these are concealed in between the two subunits of the dimer.
Thus, it is expected that the dimerization of EhODC may be
responsible for protecting EhODC enzyme from proteolytic
Mutational analysis of dimer interface residues
Molecular model of the EhODC dimer evidently shows that the
conserved catalytic residues from both monomeric subunits form
two equivalent active sites at the dimer interface (Figure 2,
Figure 9). Consequently, it can be hypothesized that the dimeric
state of EhODC enzyme is the active form. Therefore, 3D
structure based site-directed mutagenesis approach was used to
examine the functional role of EhODC dimerization. Conserved
residues of the catalytic pocket present at the dimer interface and
also the conserved residues of the dimerization interface were
The conserved catalytic residues Lys57 and Cys334 present in
the active site were selected for mutational studies, because the
structure model of EhODC as well as the sequence alignment of
Figure 11. Schematic representation of homodimers and heterodimer in the mixture of EhODC Cys334Ala and Lys57Ala mutants.
(A–C) Homodimer formation of wild-type and mutants of EhODC in individual solutions. (D) Possible combinations of EhODC monomeric subunits in
the mixture of Cys334Ala and Lys57Ala mutants forming heterodimer and homodimers.
Evidence for Functional Dimeric Form of EhODC
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EhODC with human ODC revealed that Lys57 of one subunit
(Lys69 in human) and Cys3349 of other subunit (Cys360 in
human) jointly play critical role in catalysis and substrate
specificity in a single active site pocket (Figure 2, Figure 9) [53–
55]. The residue Lys57 plays crucial role in PLP binding by
forming Schiff base to aldehyde group with its –NH2group, thus
serves as a proton donor during catalysis . The interaction of
Lys57 with PLP governs its position and correct orientation at
active site. Gel filtration analysis indicates that K57A mutant exists
in the dimeric form indicating that this mutation does not disrupt
dimerization (data not shown). However, when enzyme activity
was examined, K57A mutation was found to abolish enzyme
activity with ,2% activity as compared to the wild type
Moreover, Cys residue in the same active site from other
subunit in the active site is involved in substrate binding and
stabilizes the quinonoid intermediate by using its carbonyl group
[54,57]. This residue is crucial for decarboxylation of L-ornithine
and release of decarboxylated product towards the interface to exit
from active site. The C334A mutant was also found to be a dimer
indicating that mutation does not affect dimerization (data not
shown). However, C334A was also found to be inactive with ,2%
enzymatic activity as compared to wild type (Figure 10).
Interestingly, when the two mutant proteins K57A and C334A
were mixed in equal concentration, the enzyme activity was
partially regained having 29% activity as compared to wild type
(Figure 10). The recovery of enzyme activity on mixing these two
mutants is only possible when the two mutants associate to form a
Figure 12. Gel filtration analysis of interface residue mutants. (A) Gel-filtration chromatogram of Gly361Tyr mutant showing partial
dissociation of dimers into monomers; (B) Gel-filtration chromatogram of Lys157Ala mutant showing partial dimeric disruption.
Evidence for Functional Dimeric Form of EhODC
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heterodimer. The formation of heterodimer is anticipated to
restore one of the two active sites at the dimer interface as depicted
in figure 11. Three types of enzyme population are expected in
mutant mixture i.e. homodimers of K57A, homodimers of C334A
and heterodimers of K57A and C334A. Therefore, restoration of
approximately one-third of the wild-type enzyme activity in the
mixture of mutants is due to the dimerization of K57A and C334A
which possesses a catalytically active site pocket at one end of the
heterodimer. These mutagenesis results evidently demonstrate that
dimeric state is the functional form of ODC enzyme in E.
In mouse, 19 conserved residues at the dimer interface were
mutated to identify the key residues responsible for dimerization
. It was noted that substitution of conserved Gly387 to any
amino acid except alanine abolished the enzymatic activity. The
same result is also observed in case of Lactobacillus and hamster,
where the corresponding glycine was mutated to any bulky amino
acid resulted in inactivation of the enzyme [58,59]. Crystal
structure of mouse ODC revealed that this mutation could
position b/a-barrel at a different angle to b-sheet so that in the
mutant protein these domains have different orientations in the
dimer compared to the wild type which makes the enzyme inactive
. In the present study, EhODC Gly361 (Gly387 in mouse) was
mutated to bulky Tyr residue and its influence on dimerization
was assessed by gel filtration analysis. The chromatogram showed
partial destabilization of dimer with two distinct peaks corre-
sponding to the molecular weight of monomer and dimer
(Figure 12). The examination of enzyme activity showed that the
Gly361Tyr mutant is functionally inactive (Figure 10). These
results suggest that Gly361 in EhODC is not involved in direct
interaction between the two subunits of dimer, however it plays an
indirect role in the dimer stability through long range molecular
Additionally in the structure model and sequence alignment
analysis, Lys157 of EhODC is conserved and forms a salt bridge
with Asp3389 connecting the two monomeric subunits. At the
same position in the crystal structure of human ODC, Lys169 of
one subunit is involved in the salt bridge formation with Asp3649
of other subunit near the active site [21,22]. Thus, Lys157 of
EhODC plays a critical role in spatial arrangement of active site
residues from both the subunits in a proper orientation along with
its role in dimer formation. Mutation of Lys157 to Ala (K157A)
leads to inactivation of enzyme (Figure 10). Moreover, partial
disruption of the dimer as compared to the wild type protein was
observed for K157A mutant, because a peak corresponding to the
monomeric state of EhODC along with the dimer peak was
observed in the gel filtration chromatogram (Figure 12). These
results suggest that Lys157 plays a direct role in dimerization that
eventually leads to the active site formation.
Furthermore, a double mutant of EhODC having two mutations
i.e. G361Y and K157A was expressed in E. coli. The protein was
over-expressed using high IPTG concentration of ,2 M for
induction. This double mutant was found to be unstable and
susceptible to protease degradation during purification. Therefore,
it could not be purified for further analysis. The instability of the
double mutant G361Y and K157A could be due the dimer
disruption making the protein insoluble as well as proteolytically
Our current study, evidently demonstrates that EhODC enzyme
exists in the dimeric form. The role of dimerization with respect to
functionality was investigated by comparative structure modeling
and mutational studies. Molecular structure reveals a sharp
complementary arrangement of interface and active site residues
to support the proper spatial arrangement. Thus, it contributes
both the subunits in generation of two equivalent active sites. The
partial recovery of the enzyme activity on mixing the two mutants,
C334A and K57A which were individually inactive, shows that
dimer is the active form of EhODC. Additionally, a single
substitution at G361Y resulted in partial destabilization of the
dimer and renders the enzyme inactive. Further, K157A mutation
expected to disrupt a salt bridge K157-D3389 between two
subunits didn’t completely disrupt the dimer but inactivates the
enzyme. These results signify that various long and short range
forces play a crucial role in the dimerization and the geometry of
the dimer interface is ideal for enzyme activity. Based on these
observations, it can be proposed that disruption of functional
EhODC dimer could be a novel target for anti-amoebiasis drugs.
Molecular 3D model of EhODC dimer may support and open
possibilities to find new structure based inhibitor molecules for the
The authors thank Macromolecular Crystallographic Unit (MCU), IIT
Roorkee, for providing protein purification and computational facilities.
Conceived and designed the experiments: P RM S. Tomar. Performed the
experiments: P S. Tapas. Analyzed the data: P S. Tapas PK S. Tomar.
Contributed reagents/materials/analysis tools: PK RM S. Tomar. Wrote
the paper: P S. Tapas S. Tomar.
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www.plosntds.org 15 February 2012 | Volume 6 | Issue 2 | e1559