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

Schematic representation of dimer interface and active site of <i>Eh</i>ODC.

<p>(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 90° 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.</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

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

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

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

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