Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica.
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 oblig...
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ftdoajarticles:oai:doaj.org/article:4faba332ff6c48d39f149c003e242f99 2023-05-15T15:16:04+02:00 Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. Preeti Satya Tapas Pravindra Kumar Rentala Madhubala Shailly Tomar 2012-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0001559 https://doaj.org/article/4faba332ff6c48d39f149c003e242f99 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3289617?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0001559 https://doaj.org/article/4faba332ff6c48d39f149c003e242f99 PLoS Neglected Tropical Diseases, Vol 6, Iss 2, p e1559 (2012) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2012 ftdoajarticles https://doi.org/10.1371/journal.pntd.0001559 2022-12-31T11:14:44Z 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 ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 6 2 e1559 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Preeti Satya Tapas Pravindra Kumar Rentala Madhubala Shailly Tomar Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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 ... |
format |
Article in Journal/Newspaper |
author |
Preeti Satya Tapas Pravindra Kumar Rentala Madhubala Shailly Tomar |
author_facet |
Preeti Satya Tapas Pravindra Kumar Rentala Madhubala Shailly Tomar |
author_sort |
Preeti |
title |
Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
title_short |
Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
title_full |
Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
title_fullStr |
Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
title_full_unstemmed |
Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica. |
title_sort |
biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from entamoeba histolytica. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2012 |
url |
https://doi.org/10.1371/journal.pntd.0001559 https://doaj.org/article/4faba332ff6c48d39f149c003e242f99 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 6, Iss 2, p e1559 (2012) |
op_relation |
http://europepmc.org/articles/PMC3289617?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0001559 https://doaj.org/article/4faba332ff6c48d39f149c003e242f99 |
op_doi |
https://doi.org/10.1371/journal.pntd.0001559 |
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PLoS Neglected Tropical Diseases |
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6 |
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2 |
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e1559 |
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1766346374610157568 |