Crystal structure of <i>Eh</i>ODC monomeric subunit.

<p><b>A</b>) Cartoon diagram of the monomer showing arrangement of barrel and sheet domain. <b>B</b>) Topology diagram of monomer of <i>Eh</i>ODC where helices are represented with cylinder and sheets with the arrows connected with loops, dashed line indicates the sequence missing in the structure.</p

Tetrameric structure with dimer-dimer interaction.

<p><b>A–C</b>) shows the interaction between chain A and chain C. <b>B–D</b>) indicates the interaction between chain B and chain D. Pink dashes shows the interaction of residues through water molecule and green dashes indicates the polar interactions. Symbol (″) and (′) denotes the residues of chain C and chain D, respectively.</p

Superimposition of active site of <i>Eh</i>ODC with <i>Tb</i>ODC bound to DFMO.

<p>Residues of active site at the dimer interface are represented in sticks. <i>Tb</i>ODC residues are colored with green, <i>Eh</i>ODC residues are colored with orange. PLP and DFMO are colored with blue and polar interactions were indicated by black dashes; water molecule in shown in red sphere. Residues with (′) symbol are of opposite monomer.</p

Schematic representation of homodimers and heterodimer in the mixture of <i>Eh</i>ODC Cys334Ala and Lys57Ala mutants.

<p>(A–C) Homodimer formation of wild-type and mutants of <i>Eh</i>ODC in individual solutions. (D) Possible combinations of <i>Eh</i>ODC monomeric subunits in the mixture of Cys334Ala and Lys57Ala mutants forming heterodimer and homodimers.</p

Purification and molecular mass determination of <i>Eh</i>ODC.

<p>(A) Affinity purification of <i>Eh</i>ODC showing purified protein in 12% SDS-PAGE. Lane 1: Molecular weight marker; Lane 2: Purified <i>Eh</i>ODC-His tagged protein; Lane 3: Purified His tag cleaved protein with molecular weight ∼46 kDa. (B) Size-exclusion chromatography profile of <i>Eh</i>ODC 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 (V_o) and molecular weight (kDa) of standard proteins are indicated.</p

Sequence analysis of ODC and antizyme inhibitor, comparing the active site residues of ODC/AZI from various organisms.

<p>Abbreviation denoted: C^f for cofactor binding; B^s salt bridge formation; S substrate binding residues; I^f dimer interface residues; Dⁱ important for dimer formation. Species with the name of protein are shown on left side. <b>Colour indication:</b> Violet columns signifies the mutation in AZI; Orange columns signifies the mutated residues in <i>E. histolytica</i> ODC which are similar to AZI; Gray shows the unique mutations in <i>Eh</i>ODC which is neither conserved in ODC nor in AZI; Blue indicate the mutation in <i>Eh</i>ODC which are rarely found in AZI and functional ODC; Olive color point out the mutations in <i>Eh</i>ODC which are similar to some ODC. Sequence analysis and numbering has been done according to <i>Eh</i>ODC. Residues which are not conserved are shown by single letter, the conserved residues are indicted by – and Δ indicates the deleted amino acids. % identity indicates the identity of <i>Eh</i>ODC sequence with other homologous ODC sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053397#pone.0053397-Ivanov1" target="_blank">[46]</a>.</p

Sequence of mutagenic primers and annealing temperature used for PCR amplification of mutant plasmids.

<p>Mutated nucleotides are underlined. S: sense and An: antisense.</p

Circular Dichroism spectroscopy of <i>Eh</i>ODC.

<p>A Far-UV CD spectrum of 0.35 mg/ml <i>Eh</i>ODC. 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.</p

Oligomeric state determination.

<p>MALDI-TOF MS analysis of <i>Eh</i>ODC showing two peaks corresponding to ∼44558.430 Da and ∼90667.295 Da. The insert shows 12% SDS-PAGE analysis of glutaraldehyde crosslinked <i>Eh</i>ODC. 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 <i>Eh</i>ODC; Lane 4: Purified protein not treated with glutaraldehyde.</p

Phylogenetic relationship of <i>Eh</i>ODC with antizyme inhibitor and ODC.

<p>Sequence of ODC and their evolutionary related homologous were retrieved from various sources. <b>Antizyme inhibitor</b> of <i>Homo sapiens</i> (BAA23593.1), <i>Nomascus leucogenys</i> (XP_003256127.1), <i>Macaca mulatta</i> (XP_002805501.1), <i>Mus musculus</i> (AAB87464.1), <i>Rattus norvegicus</i> (BAA23594.1), <i>Monodelphis domestica</i> (XP_001369332.1), <i>Xenopus laevis</i> (NP_001087584.1), <i>Danio rerio</i> (BAB84695.1), <i>Tetraodon nigroviridis</i> (ENSTNIT00000008148.1), <i>Anolis carolinensis</i> (XP_003219500.1), <i>Gallus gallus</i> (NP_001008729.1), <i>Ornithorhynchus anatinus</i> (XP_001506230.1), <i>Canis familiaris</i> (XP_849306.1), <i>Bos Taurus</i> (NP_001076080.1), <i>Loxodonta africana</i> (XP_003408472.1). <b>Ornithine decarboxylase sequence from </b><i>Aedes aegypti</i> (EAT48998.1), <i>Entamoeba histolytica</i> (AAX35675.1), <i>Plasmodium falciparum</i> (AAF14518.1), <i>Leishmania donovani</i> (AAA29259.1), <i>Datura stramonium</i> (CAA61121.1), <i>Solanum lycopersicum</i> (NP_001234616.1), <i>Glycine max</i> (CAD91349.1), <i>Chlamydomonas reinhardtii</i> (CAE46409.1), <i>Monodelphis domestica</i> (XP_001371947.1), <i>Bos Taurus</i> (AAA92339.1), <i>Macaca mulatta</i> (NP_001185615.1), <i>Homo sapiens</i> (NP_002530.1), <i>Mus musculus</i> (NP_038642.2), <i>Anolis carolinensis</i> (XP_003215471.1), <i>Trypanosoma brucei</i> (AAA30219.1), <i>Xenopus laevis</i> (CAA39760.1).</p