Disease relevance of Tb11.01.5300

• Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei [1].
• 26 patients with arseno-resistant Trypanosoma brucei gambiense trypanosomiasis were treated with difluoromethylornithine (eflornithine), an inhibitor of ornithine decarboxylase, given intravenously, then orally [2].
• High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase [3].
• A mouse model of T. brucei infection was used to determine whether the mutant was still infective and was found to develop either extremely low or undetectable levels of parasitemia, suggesting that in T. brucei, ODC activity is essential for establishing an infection [4].
• Ornithine decarboxylase in Trypanosoma brucei brucei: evidence for selective toxicity of difluoromethylornithine [5].

High impact information on Tb11.01.5300

• However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the delta-amino group of ornithine analogs in the x-ray structures of ODC [6].
• The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating l-ornithine [6].
• Mutational analysis was carried out to determine whether the Asp-to-Glu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity [6].
• Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the rate-determining step in the biosynthesis of polyamines [7].
• X-ray structure determination of Trypanosoma brucei ornithine decarboxylase bound to D-ornithine and to G418: insights into substrate binding and ODC conformational flexibility [7].

Biological context of Tb11.01.5300

• Glu-274 was previously demonstrated to play a direct role in carbanion stabilization, and thus the large carbon isotope effect (k12/k13 = 1.055) is consistent with an impaired rate of decarboxylation compared to wild-type ODC [8].
• Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association [9].
• We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities [10].
• With our first stable transfection vector, we replaced the 2.8 kb SacII ODC gene-containing fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter [11].
• The resistant phenotype was stable in the absence of DFMO, in which state there was no increase in ODC abundance or activity [12].

Anatomical context of Tb11.01.5300

• The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines [13].
• In the present study we demonstrate that C. fasciculata ODC is rapidly degraded also in mammalian systems like CHO cells and rabbit reticulocyte lysate, suggesting that the degradation signals of the enzyme are recognised by the mammalian systems [14].

Associations of Tb11.01.5300 with chemical compounds

• In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite [15].
• The two proteins, TbMRPA and TbMRPE, were each overexpressed in trypanosomes, with or without co-expression of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma-glutamyl-cysteine synthetase [16].
• Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines [17].
• Alterations in ornithine decarboxylase characteristics account for tolerance of Trypanosoma brucei rhodesiense to D,L-alpha-difluoromethylornithine [18].
• The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304] [19].

Analytical, diagnostic and therapeutic context of Tb11.01.5300

• The sequence analysis revealed a high homology between C. fasciculata ODC and L. donovani ODC, despite the difference in stability [20].
• The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8 [19].
• The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy [19].
• Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei [21].
• The two ODC gene knockout lines were verified by Southern and Northern hybridization, and confirmed by Western blot and enzymatic activity assay [11].

References