Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica

Abstract

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 α-helix, β-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.

Figure 1. The enzymatic reaction catalyzed by ornithine decarboxylase.

The pyridoxal phosphate (PLP)-dependent ODC enzyme catalyzes decarboxylation of ornithine and produces putrescine.

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.

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.

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 (V_o) and molecular weight (kDa) of standard proteins are indicated.

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 glutaraldehyde 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 glutaraldehyde.

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.

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, β/α-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.

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 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.

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.

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.

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.