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Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.

ABSTRACT: The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica that is responsible for the biosynthesis of prostaglandin (PG)E2. PGE2 produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE2 but not into PGD2 or PGF2alpha. COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors. Taken together, these studies reveal that E. histolytica produces PGE2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.

SUBMITTER: Dey I

PROVIDER: S-EPMC263853 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.

Dey Indranil I Keller Kathy K Belley Adam A Chadee Kris K

Proceedings of the National Academy of Sciences of the United States of America 20031029 23

The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica that is responsible for the biosynthesis of prostaglandin (PG)E2. PGE2 produced by ameba was constitutive bu ...[more]

PMID: 14585927

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Project description:BACKGROUND: The 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 RESULTS: To 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 approximately 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 approximately 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. CONCLUSION: To 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.

Dataset Information

Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.

Publications

Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica.

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