title: Characterization of the Entamoeba histolytica ornithine decarboxylase-like enzyme.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Anupam
authors.initials: A
authors.lastname: Jhingran
authors.forename: Prasad K
authors.initials: PK
authors.lastname: Padmanabhan
authors.forename: Sushma
authors.initials: S
authors.lastname: Singh
authors.forename: Krishanpal
authors.initials: K
authors.lastname: Anamika
authors.forename: Abhijeet A
authors.initials: AA
authors.lastname: Bakre
authors.forename: Sudha
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Alok
authors.initials: A
authors.lastname: Bhattacharya
authors.forename: Narayanaswamy
authors.initials: N
authors.lastname: Srinivasan
authors.forename: Rentala
authors.initials: R
authors.lastname: Madhubala
mesh_terms: D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D000518:Eflornithine / Q000494:pharmacology; D016521:Electrophoresis, Gel, Pulsed-Field; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009952:Ornithine / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011073:Polyamines / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D011073:Polyamines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D009952:Ornithine; D009955:Ornithine Decarboxylase; D000518:Eflornithine