Research Article: Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

Date Published: January 2, 2008

Publisher: Public Library of Science

Author(s): Anupam Jhingran, Prasad K. Padmanabhan, Sushma Singh, Krishanpal Anamika, Abhijeet A. Bakre, Sudha Bhattacharya, Alok Bhattacharya, Narayanaswamy Srinivasan, Rentala Madhubala, Anuradha Lohia

Abstract: BackgroundThe polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme.Methodology and ResultsTo use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of ∼1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size ∼46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids—aspartate-332, aspartate-361, and tyrosine-323—by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity.ConclusionTo our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

Partial Text: Entamoeba histolytica is a unicellular protozoan parasite that infects about 50 million people each year and may cause potentially life-threatening diseases such as hemorrhagic colitis and/or extraintestinal abscesses [1]. The infections are primarily treated by antiamoebic therapy. Drugs of choice for invasive amoebiasis are tissue-active agents such as metronidazole, tinidazole, and chloroquine [2]. Although drug resistance to E. histolytica does not appear to be a serious problem, there are occasional reports of failure with metronidazole suggesting the possibility of development of clinical drug resistance [3].

The polyamines putrescine, spermidine, and spermine are polycationic organic compounds present in all eukaryotic cells, including parasitic protozoans. It was reported earlier that the polyamines are essential for the proliferation of normal cells and for differentiation [6].



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