Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Arteaga-Nieto, Pablo; López-Romero, Everardo; Terán-Figueroa, Yolanda; Cano-Canchola, Carmen; Arias, J.; Flores-Carreón, Arturo; Calvo-Méndez, Carlos

doi:10.1016/s0014-4894(02)00137-6

Cited by 11 publications

(16 citation statements)

References 31 publications

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“…Previously, it has been reported that the Eh ODC enzyme exists only as a homopentamer [25]. However, our studies evidently demonstrate that Eh ODC is functionally active in the dimeric form.…”

Section: Introductioncontrasting

confidence: 46%

“…ODC purified from E. histolytica has previously been reported to exist in a pentameric state [25]. Three dimensional crystal structure studies of ODCs from different sources have shown that the enzyme exists as a homodimer and association of monomeric subunits directs the formation of two equivalent catalytic pockets at the dimer interface.…”

Section: Resultsmentioning

confidence: 99%

“…In E. histolytica , the only enzyme of polyamine biosynthesis reported to exist is ODC. E. histolytica ODC ( Eh ODC) has been reported to form homopentamers [25]. Interestingly, Eh ODC is insensitive to DFMO and DFMO has no inhibitory effect on the cell growth of the parasite [25]–[27].…”

Section: Introductionmentioning

confidence: 99%

“…E. histolytica ODC ( Eh ODC) has been reported to form homopentamers [25]. Interestingly, Eh ODC is insensitive to DFMO and DFMO has no inhibitory effect on the cell growth of the parasite [25]–[27]. Therefore, it is necessary to develop an alternate method for inhibition of Eh ODC enzyme for targeting the polyamine biosynthetic pathway to curb the disease.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

et al. 2012

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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 ResultsIn 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.ConclusionTo 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.

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Section: Introductioncontrasting

confidence: 46%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

et al. 2012

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“…Interestingly, DFMO being an effective drug against T. brucei gambiense is reported to have relatively poor effect on the more virulent strain T. brucei rhodesiense [32],[33]. Furthermore, the ODC of E. histolytica , being a pathogenic strain from protozoa kingdom, is insensitive to DFMO due to sequence divergence in the substrate binding residues [29],[34],[35],[36]. Natural resistance to DFMO within the same Trypanosome species as well as within the protozoa kingdom draws attention towards the sequence and structural divergence for their evolutionary adaptation.…”

Section: Introductionmentioning

confidence: 99%

Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution

et al. 2013

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BackgroundPolyamine 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 FindingThe 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 P212121 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/SignificanceWe 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.

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PLP-dependent enzymes as potential drug targets for protozoan diseases

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et al. 2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Cited by 11 publications

References 31 publications

Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution

PLP-dependent enzymes as potential drug targets for protozoan diseases

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