Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.17 (ornithine decarboxylase)
 6,351 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rapid, polyamine-induced degradation of mammalian ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC) is though to be controlled by the availability of a small, ODC-binding protein termed antizyme. In this study we have investigated the ability of antizyme to bind ODC protein in various altered physiological states. In particular, cold, NaCl, spermidine and deprivation of coenzyme and substrate enhance enzyme-antizyme complex formation and are all found to promote ODC homodimer dissociation. Conversely, conditions that maintain the active ODC homodimer state prevent antizyme binding and inactivation of ODC. Further, covalent modification of ODC near its active site by difluoromethylornithine or phosphate also increases its sensitivity to antizyme. These results suggest that the initial signal in ODC degradation may actually be a subtle conformational change in the enzyme that enables antizyme to bind to the enzyme and may subsequently facilitate its degradation.
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PMID:Conformational changes in ornithine decarboxylase enable recognition by antizyme. 210 55

The mechanism of action of tumor promoters may involve the modulation of gene expression, e.g., the induction of ornithine decarboxylase (ODC). The tumor promoter phorbol-13-myristate-12-acetate (PMA) induces chromosomal damage via the intermediacy of active oxygen species which may trigger the activation of certain genes. Therefore, we have studied the effect of antioxidants on the induction of ODC by PMA, medium change only and medium change plus PMA in mouse mammary tumor cells Mm5mt/C1. CuZn-superoxide dismutase (SOD, a scavenger of superoxide radicals), catalase (CAT, a scavenger of hydrogen peroxide) and mannitol (a scavenger of hydroxyl radicals) suppressed ODC induction under all three conditions. The relative inhibitory potency of the antioxidants was always SOD less than CAT less than mannitol less than SOD + CAT. Maximal suppression by SOD + CAT was approximately 50%. It is concluded that active oxygen species play a role in ODC induction by factors contained in serum and by PMA.
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PMID:The induction of ornithine decarboxylase by phorbol 12-myristate 13-acetate or by serum is inhibited by antioxidants. 664 Aug 44

Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.
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PMID:Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis. 774 28

In a PCR with degenerate primers encoding highly conserved amino acids within ornithine decarboxylases (ODCs) of several organisms, a fragment of the ODC gene of the free-living nematode Panagrellus redivivus was isolated. Northern blot analysis revealed a single 1.7 kb transcript in a mixed-stage population of animals. From this RNA source, a cDNA library was constructed and screened with the PCR fragment. Several cDNA clones were isolated, one of which encodes the complete 435-amino-acid ODC enzyme with a calculated molecular mass of 47.1 kDa. The P. redivivus ODC possesses 126 of the 136 highly conserved amino acids in the enzymes from fungi, invertebrates and vertebrates. Functional amino acids are conserved, suggesting that the two active sites of the P. redivivus ODC are formed at the interface of a homodimer, as described for mammalian ODCs.
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PMID:Molecular cloning and characterization of ornithine decarboxylase cDNA of the nematode Panagrellus redivivus. 777 52

In its active form mammalian ornithine decarboxylase (ODC) is a homodimer composed of two 53-kDa subunits while the monomer retains no enzymic activity. In the present study we demonstrate that Gly387 of mouse ODC plays an important role in enabling dimer formation. Gly387 of mouse ODC, an evolutionary conserved residue, was converted to all possible 19 amino acids using site-directed mutagenesis. With the exception of alanine, all other substitutions of Gly387 completely abolished enzymic activity. Cross-linking analysis and fractionation through a Superose-12 sizing column have demonstrated that mutant subunits are detected only in their monomeric form. These results strongly suggest that the primary lesion of substitution at position 387 of mouse ODC is the inability of mutant subunits to associate with each other to form the active homodimers. In agreement with this conclusion, G387A, the only mutant that retained partial activity, displayed reduced dimerization. The degradation rate of ODC mutants in which Gly387 was substituted by aspartic acid or alanine was enhanced compared to the wild-type enzyme, suggesting that monomers may be more susceptible to degradation.
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PMID:Gly387 of murine ornithine decarboxylase is essential for the formation of stable homodimers. 824 70

The active form of mammalian ornithine decarboxylase (ODC) is a homodimer consisting of two monomer subunits of 53 kDa each. We have used in vitro hybridization of two different catalytically inactive mutants of ODC to determine whether in the wild-type enzyme each monomer contains an independent active site or whether the active sites are shared at the interfaces between the two subunits. Two distinct mutants were obtained using oligonucleotide-directed mutagenesis: In one, cysteine-360, the major alpha-(difluoromethyl)ornithine (alpha-DFMO, a suicide inhibitor of ODC) binding site was converted to alanine. In the other, lysine-69, the pyridoxal 5'-phosphate (PLP, the cofactor of ODC) binding residue was converted to alanine. Expression of each mutant, in vitro, in reticulocyte lysate translation mix, results in the production of a completely inactive enzyme. In contrast, their coexpression restores enzymatic activity to about 25% of the wild-type enzyme. Moreover, coexpression of wild-type subunits with monomers containing both inactivating mutations reduced their activity to about 25%, while their coexpression with monomers that contain a single inactivating mutation reduced the activity to 50%. Cross-linking analysis has demonstrated that activity restoration and repression are both fully correlated with the formation of heterodimers between mutant subunits and between mutant and wild-type subunits, respectively. We therefore conclude that the active site of ODC is formed at the interface of the two monomers through the interaction of the cysteine-360-containing region of one monomer subunit with the region that contains lysine-69 of the other subunit.
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PMID:Intersubunit location of the active site of mammalian ornithine decarboxylase as determined by hybridization of site-directed mutants. 850 4

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. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.
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PMID:Altering the reaction specificity of eukaryotic ornithine decarboxylase. 1098 70

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.
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PMID:Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity. 1157 38

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.
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PMID:Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function. 1168 31

The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal alpha/beta-barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon alpha-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structure-based approach for the design of novel malaria-specific inhibitors.
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PMID:Comparative properties of a three-dimensional model of Plasmodium falciparum ornithine decarboxylase. 1255 88


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