Structural insight into DFMO resistant ornithine decarboxylase from Entamoeba histolytica: an inkling to adaptive evolution

Abstract

Background: Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.

Methodology/principal finding: The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P2(1)2(1)2(1) symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.

Conclusions/significance: We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.

Figure 1. Purification and gel filtration profile of EhODCΔ15.

A) 12% SDS-PAGE gel showing the affinity purified protein Lane 1: Molecular weight markers shown in kDa. Lane 2–3: Protein purified by affinity chromatography. B) Elution profile of the EhODC1Δ15 protein. The protein was eluted at a volume of 74 ml corresponding to molecule weight of ∼87 kDa. Insert shows the purified protein in 12% SDS-PAGE after gel filtration chromatography.

Figure 2. Schematic representation of overall structure of the model obtained after molecular replacement.

A) Cartoon diagram of tetrameric model of EhODC showing AB-CD, dimer-dimer interface; B) Active site of EhODC at the interface of dimer where (′) denotes the residues from the other subunit.

Figure 3. Tetrameric structure with dimer-dimer interaction.

A–C) shows the interaction between chain A and chain C. B–D) 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.

Figure 4. Crystal structure of EhODC monomeric subunit.

A) Cartoon diagram of the monomer showing arrangement of barrel and sheet domain. B) Topology diagram of monomer of EhODC where helices are represented with cylinder and sheets with the arrows connected with loops, dashed line indicates the sequence missing in the structure.

Figure 5. Superimposition of active site of EhODC with TbODC bound to DFMO.

Residues of active site at the dimer interface are represented in sticks. TbODC residues are colored with green, EhODC 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.

Figure 6. Multiple sequence alignment of ornithine decarboxylase and its homologues antizyme inhibitor to determine the conservation of sequence and mutation of active site and substrate binding residues.

Circles indicate the residues important for enzymatic activity. Numbering is according to EhODC.

Figure 7. Sequence analysis of ODC and antizyme inhibitor, comparing the active site residues of ODC/AZI from various organisms.

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. Colour indication: Violet columns signifies the mutation in AZI; Orange columns signifies the mutated residues in E. histolytica ODC which are similar to AZI; Gray shows the unique mutations in EhODC which is neither conserved in ODC nor in AZI; Blue indicate the mutation in EhODC which are rarely found in AZI and functional ODC; Olive color point out the mutations in EhODC which are similar to some ODC. Sequence analysis and numbering has been done according to EhODC. 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 EhODC sequence with other homologous ODC sequences .

Figure 8. Active sites comparison of functional ODC, antizyme inhibitor and EhODC.

A) Human ODC active site residues colored in blue. B) EhODC active site residues identical to human ODC colored blue, residues identical to AZI colored green and unique to EhODC colored red. C) AZI interface region showing residues identical to human ODC in blue and those are mutated colored green.

Figure 9. Phylogenetic relationship of EhODC with antizyme inhibitor and ODC.

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