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

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.
 ...
PMID:Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine. 1173 57

Glutamine (GLN) and arginine (ARG) are recognized for their ability to modulate immune cell function. However, the metabolic pathways involved in their action remain unclear. It was recently shown that GLN- or ARG-enriched diets increased radical oxygen species (ROS) production by neutrophils from stressed rats. Since these two amino acids have a tied metabolism, we hypothesized that conversion between GLN and ARG (and its active metabolites NO* and polyamines) might be involved. To test this hypothesis male Sprague-Dawley rats (n 117) were randomized into thirteen groups: rats in eleven groups were rendered catabolic by dexamethasone injection (1.5 mg/kg per d for 5 d) and 6.8 mmol either GLN, ARG or non-essential amino acids (NEAA; glycine, alanine and histidine)/kg per d were given by the enteral route; one group was pair-fed to the treated groups. The regimens of all the groups were rendered isonitrogenous by the addition of NEAA. The last group was not treated and was fed ad libitum. For each supplementation three subgroups were formed, each of which received a specific inhibitor: methionine sulfoximine (inhibitor of GLN synthase; 100 mg/kg per d), S-methylthiourea (inhibitor of inducible NO* synthase (iNOS); 50 mg/kg per d) and difluoromethylornithine (inhibitor of ornithine decarboxylase (ODC); 50 mg/kg per d). Oxidative metabolism, intracellular H2O2, and extracellular O2*- production were measured in unstimulated and phorbol myristate acetate-stimulated polymorphonuclear neutrophils. GLN- and ARG-enriched diets increased respiratory burst by neutrophils (oxidative metabolism of 152 (sem 24) and 138 (sem 45) v. 57 (sem 18) mV for GLN-, ARG- and NEAA-enriched diets respectively, P<0.05). In vivo inhibition of iNOS or ODC decreased ROS production induced by GLN and ARG. In vivo inhibition of GLN synthase did not modify the effect of ARG on ROS production. In conclusion, GLN and ARG modulate ROS production in neutrophils from stressed rats by the same pathway involving polyamine and NO* synthesis.
 ...
PMID:Evidence that glutamine modulates respiratory burst in stressed rat polymorphonuclear cells through its metabolism into arginine. 1249 91

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the decarboxylation of l-Orn to putrescine, a rate-limiting step in the formation of polyamines. The X-ray crystal structures of ODC, complexed to several ligands, support a model where the substrate is oriented with the carboxyl-leaving group buried on the re face of the PLP cofactor. This binding site is composed of hydrophobic and electron-rich residues, in which Phe-397 is predicted to form a close contact. Mutation of Phe-397 to Ala reduces the steady-state rate of product formation by 150-fold. Moreover, single turnover analysis demonstrates that the rate of the decarboxylation step is decreased by 2100-fold, causing this step to replace product release as the rate-limiting step in the mutant enzyme. These data support the structural prediction that the carboxyl-leaving group is positioned to interact with Phe-397. Multiwavelength stopped-flow analysis of reaction intermediates suggests that a major product of the reaction with the mutant enzyme is pyridoximine 5'-phosphate (PMP), resulting from incorrect protonation of the decarboxylated intermediate at the C4' position. This finding was confirmed by HPLC analysis of the reaction products, demonstrating that Phe-397 also plays a role in maintaining the integrity of the reaction chemistry. The finding that the carboxylate-leaving group is oriented on the buried side of the PLP cofactor suggests that ODC facilitates decarboxylation by destabilizing the charged substrate carboxyl group in favor of an electrostatically more neutral transition state.
 ...
PMID:Ornithine decarboxylase promotes catalysis by binding the carboxylate in a buried pocket containing phenylalanine 397. 1262 59

Hexamita inflata is an amitochondriate flagellated protozoon which inhabits O(2)-limited environments. With the aid of 1H NMR spectroscopy, analysis of the metabolic fluxes in H. inflata grown in complex media under limited O(2) was performed. Almost complete carbon recovery from maltose (the principle carbohydrate source in the medium) catabolism was calculated from the measured increase in concentration of ethanol, alanine, acetate and lactate (and estimated CO(2) production). Difference spectra and amino acid analysis also identified changes in concentration of metabolites belonging to the arginine dihydrolase (ADH) pathway. The enzymes of the ADH pathway were detected in extracts with the following activities (in nmoles min(-1) x (mg of protein) x (-1)): arginine deiminase, 3.30; catabolic ornithine carbamyltransferase (OCT), 1.3; anabolic OCT, 93.0; and carbamate kinase, 1829. The organism metabolized the ornithine produced from catabolic OCT activity to putrescine via ornithine decarboxylase (ODC). The polyamines, spermidine and spermine, were formed by the sequential addition of the aminopropyl group of decarboxylated S-adenosyl-L-methionine (SAM) by the respective polyamine synthases. In addition, asparaginase activity was confirmed in H. inflata, catalysing the deamination of asparagine generating aspartate and ammonia. This study also indicates that, as with other amitochondriate protozoa and some bacteria, the ADH pathway significantly contributes to the energy yield of the cell, particularly under O(2)-limited conditions.
 ...
PMID:Bacterial-like energy metabolism in the amitochondriate protozoon Hexamita inflata. 1270 92

The cholecystokinin 2 receptor (CCK2R) increases proliferation of normal and neoplastic gastrointestinal cells and activates various mitogenic signaling pathways when stimulated by gastrin. To study the incidence of permanent activation of this receptor in tumorigenicity, a constitutively active mutant was generated by replacing residue Glu151 in the conserved E/DRY motif by Ala. Expression of the E151A-CCK2R mutant in NIH-3T3 cells causes ligand-independent activation of phospholipase C and ornithine decarboxylase, two enzymes critical for mitogenesis. Strikingly, the constitutive activity of this mutant was associated with dramatic alteration of NIH-3T3 cell morphology, enhanced cell proliferation and invasion. Moreover, injection of cells expressing E151A-CCK2R in nude mice resulted in the development of large and rapidly growing tumors. By contrast, none of these effects was observed with cells expressing the wild-type CCK2R, indicating that the tumorigenic properties of the E151A-CCK2R mutant is the result of its constitutive activation. To date, this is the first report that provides evidence for the high tumorigenic effect of a constitutively active CCK2R mutant, thus raising a potential role of the CCK2R in human cancer.
 ...
PMID:High tumorigenic potential of a constitutively active mutant of the cholecystokinin 2 receptor. 1295 87

The Epstein-Barr virus thwarts immune surveillance through a Gly-Ala repeat (GAr) within the viral Epstein-Barr virus-encoded nuclear antigen 1 protein. The GAr inhibits proteasome processing, an early step in antigen peptide presentation, but the mechanism of proteasome inhibition has been unclear. By embedding a GAr within ornithine decarboxylase, a natural proteasome substrate that does not require ubiquitin conjugation, we now demonstrate inhibition in a purified system, excluding involvement of ubiquitin conjugation or of proteins extraneous to substrate and proteasome. We show further that the GAr acts as a stop-transfer signal in proteasome substrate processing, resulting in vivo in partial proteolysis that halts just short of the GAr. Similarly, introducing a GAr into green fluorescent protein destabilized by the ornithine decarboxylase degradation domain also stops the progress of proteolysis, leading to the accumulation of partial degradation products. We postulate that the ATP motor of the proteasome slips when it encounters the GAr, impeding further insertion and, in this way, halting degradation.
 ...
PMID:Repeat sequence of Epstein-Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing. 1468 54

Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle.
 ...
PMID:Multiple active site conformations revealed by distant site mutation in ornithine decarboxylase. 1547 92

Mouse ODC (ornithine decarboxylase) is quickly degraded by the 26S proteasome in mammalian and fungal cells. Its degradation is independent of ubiquitin but requires a degradation signal composed of residues 425-461 at the ODC C-terminus, cODC (the last 37 amino acids of the ODC C-terminus). Mutational analysis of cODC revealed the presence of two essential elements in the degradation signal. The first consists of cysteine and alanine at residues 441 and 442 respectively. The second element is the C-terminus distal to residue 442; it has little or no sequence specificity, but is intolerant of insertions or deletions that alter its span. Reducing conditions, which preclude all well-characterized chemical reactions of the Cys(441) thiol, are essential for in vitro degradation. These experiments imply that the degradative function of Cys(441) does not involve its participation in chemical reaction; it, instead, functions within a structural element for recognition by the 26S proteasome.
 ...
PMID:Structural elements of the ubiquitin-independent proteasome degron of ornithine decarboxylase. 1797 31

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.
 ...
PMID:Docking of antizyme to ornithine decarboxylase and antizyme inhibitor using experimental mutant and double-mutant cycle data. 1946 28

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.
 ...
PMID:Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids. 1964 94


<< Previous 1 2 3 4 5 Next >>