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.
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.
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 dimer.