Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors.

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10-5 mM-1·s-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria.

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% ß-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.

Ornithine decarboxylase may function as an initiation factor for RNA polymerase I.

Reparts suggest that the activity of RNA polymerase I is modulated by a labile protein with a hlaf-life of 10 to 20 minutes. Ornithine decarboxylase is the only labile protein (half-life, 10 to 20 minutes) that increases in activity prior to increased RNA polymerase I activity. The addition of a small amount of a highly purified ornithine decarboxylase preparation to an RNA polymerase I assay increases the initial rate of the reaction as well as the time for which the assay is linear. The incorporation patterns of 14C-labeled adenosine triphosphate and 32P-labeled adenosine triphosphate into RNA indicate that the addition of ornithine decarboxylase to the RNA polymerase assay increases the rate of initiation. This report demonstrates a novel way to purify ornithine decarboxylase by RNA polymerase I affinity chromatography and presents data in support of the hypothesis that the labile protein which modulates RNA polymerase I activity is ornithine decarboxylase.

Characterization of a second ornithine decarboxylase isolated from Morganella morganii.

The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

Characterization of highly purified ornithine decarboxylase from rat heart.

A highly purified preparation of heart ornithine decarboxylase was obtained from isoproterenol-treated rats. The molecular and catalytic properties of the cardiac enzyme were investigated. The isoelectric point of the enzyme appeared to be 4.9, and the molecular weight was estimated to be 54000 by SDS-polyacrylamide gel electrophoresis. Under nondenaturing conditions, the molecular weight of the partially purified enzyme was 10000-110000 as determined by gel filtration, whereas a significantly lower (Mr approx. 70000) value was obtained for purified ornithine decarboxylase. Both Km for the substrate and Vmax were affected by the dithiothreitol concentration in the assay mixture. In particular, the Km for ornithine was found to be about 0.09 mM in the presence of 2.9 mM dithiothreitol and appeared to decrease at lower dithiothreitol concentrations. The Km for pyridoxal phosphate was about 0.09 microM; putrescine and lysine inhibited the enzyme competitively, with Ki values of 1.3 and 11.7 mM, respectively. The existence of two different forms of ornithine decarboxylase in cardiac tissue was indicated by DEAE-cellulose chromatography.

Purification and properties of ornithine decarboxylase from Tetrahymena pyriformis.

In Tetrahymena pyriformis, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities are present in the cytosolic and nuclear fractions and reach maximal values in the middle and late log phases of growth, respectively. The two activities have been purified to homogeneity by ammonium sulfate fractionation (20-45%), anion-exchange chromatography (DEAE-Bio-Gel A), gel-filtration (Sephadex G-150 and Sephadex G-100 superfine) and hydrophobic chromatography (Phenyl-Sepharose). Both the crude and the purified enzyme preparations are inactivated irreversibly by alpha-difluoromethylornithine, a suicide inhibitor of mammalian ornithine decarboxylase. The enzyme preparations from the nucleus and cytosol each showed a single band on polyacrylamide gel electrophoresis under native and denaturing conditions and on acrylamide gel electrofocusing. Both activities show the same pH optima (8.6) isoelectric point (5.3), molecular weight (64 000) and Kmorn (4.7 microM). The Km for L-lysine is 0.5 mM. The two activities also cross-react with acidic antizyme extracted from E. coli mutant MA 255. Based on the physicochemical properties, one can safely conclude that cytosolic and nuclear activities reside on the same protein molecule.

Physical and kinetic distinction of two ornithine decarboxylase forms in Physarum.

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) can be isolated from crude plasmodial homogenates of Physarum polycephalum. Both forms catalyze the stoichiometric production of putrescine and CO2 from ornithine, yet they are distinguished by (a) a large difference in their affinity for coenzyme (apparent Km values of 0.13 and 33 muM); (b) a differential stability to extended dialysis of crude homogenates at 4 degrees C; and (c) the tendency of the low affinity form to polymerize when suspended in low ionic strength borate and phosphate buffers. These forms appear to be alternate states of a basic catalytic subunit in that (a) they both demonstrate monomer and dimer molecular forms of 80 000 and 160 000 daltons, respectively, depending on the buffer content; (b) they coelute from DEAE-Cellulose ion-exchange columns; and (c) they vary in activity in approximately equivalent yet opposite directions in response to factors which alter this organism's growth or metabolism. These data suggest that ornithine decarboxylase activity may be modulated by the control of the transition of this enzyme between the active and the relatively less active form.

The regulation of mouse liver ornithine decarboxylase by metabolites.

The enzyme ornithine decarboxylase (L-Ornithine carboxy-lyase, EC 4.1.1.17), has been partially purified from the livers of mice subjected to partial hepatectomy (6-8 h previously). Mouse liver ornithine decarboxylase requires pyridoxal phosphate, and dithiothreitol for maximal activity. The enzyme has a pH optimum of 7.3, it is inhibited in the presence of 0.3 M phosphate, glycine, Tricine and Tris. It shows no dependence on metal ions and is inhibited by high salt concentrations, particularly ammonium salts. The kinetics of the enzyme have been studied with putrescine (and analogs), spermidine and spermine, in the presence of both high and low levels of pyridoxal phosphate. High concentrations of pyridoxal phosphate inhibit the enzyme. The enzyme is also inhibited by low concentrations of putrescine (1 mM). As the concentration of putrescine increased to 10 mM, non-competitive inhibition was observed, this could be reversed by addition of higher levels of pyridoxal phosphate. Spermidine and spermine inhibit (noncompetitively) only at high concentrations (10 mM). Ornithine inhibits at high concentrations (2 mM). Spectral studies have shown that the observed kinetics of competitive inhibition at low concentrations of polyamine changing to noncompetitive inhibition at high polyamine concentrations are due to competition between enzyme and substrate (or inhibitor) for free (non-enzyme bound) pyridoxal phosphate. Noncompetitive inhibition arises through the formation of transient Schiff base complexes between amines and free pyridoxal phosphate. It also appears that the binding of substrate to the active site takes place through Schiff base formation with enzyme bound pyridoxal phosphate.

Purification and some properties of ornithine decarboxylase from rat liver.

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeneity from the livers of thioacetamide- and DL-alpha-hydrazino-delta aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5.10(5)-fold with an 8% yield; the specific activity of the final enzyme preparation was 1.1.10(6) nmol CO2/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50000 on SDS-polyacrylamide gels. The isoelectric point of the enzyme was pH 4.1.

Regulation of the Escherichia coli biosynthetic ornithine decarboxylase activity by phosphorylation and nucleotides.

A protein kinase which phosphorylates in vitro the biosynthetic ornithine decarboxylase (ODC) was partially purified from Escherichia coli. In vivo phosphorylation of ODC occurs after incubation of E. coli with [32P]orthophosphate. When the recombinant ODC was incubated with calf intestine alkaline phosphatase it was inactivated and this inactive ODC could be reversibly activated allosterically only by guanyl or uracyl phosphate analogues at a concentration of 10(-4) or 10(-3) M. The pH optimum of the [8-3H]GTP binding was determined and it was shown that the GTP binding is proportional to the amount of ODC. The [8-3H]GTP binds specifically to ODC as was proved by the addition of cold GTP or ATP. High concentration of GTP can dissociate the ODC-antizyme complex and either reactivate or liberate the ODC. Therefore, a working hypothesis is suggested describing the regulation of ODC by phosphorylation-dephosphorylation reaction or by antizyme and nucleotide analogues interaction.

Leishmania donovani: impairment of the cellular immune response against recombinant ornithine decarboxylase protein as a possible evasion strategy of Leishmania in visceral leishmaniasis.

Ornithine decarboxylase, the rate limiting enzyme of the polyamine biosynthesis pathway, is significant in the synthesis of trypanothione, T(SH)2, the major reduced thiol which is responsible for the modulation of the immune response and pathogenesis in visceral leishmaniasis. Data on the relationship between ornithine decarboxylase and the cellular immune response in VL patients are limited. Therefore, we purified a recombinant ornithine decarboxylase from Leishmania donovani (r-LdODC) of approximately 77kDa and examined its effects on the immunological responses in peripheral blood mononuclear cells of human visceral leishmaniasis cases. For these studies, α-difluoromethylornithine was tested as an inhibitor and was used in parallel in all experiments. The r-LdODC was identified as having a direct correlation with parasite growth and significantly increased the number of promastigotes as well as axenic amastigotes after 96h of culture. The stimulation of peripheral blood mononuclear cells with r-LdODC up-regulated IL-10 production but not IFN-γ production from CD4(+) T cells in active as well as cured visceral leishmaniasis cases, indicating a pivotal role for r-LdODC in causing strong immune suppression in a susceptible host. In addition, severe hindrance of the immune response and anti-leishmanial macrophage function due to r-LdODC, as revealed by decreased IL-12 and nitric oxide production, and down-regulation in mean fluorescence intensities of reactive oxygen species, occurred in visceral leishmaniasis patients. There was little impact of r-LdODC in the killing of L. donovani amastigotes in macrophages of visceral leishmaniasis patients. In contrast, when cultures of promastigotes and amastigotes, and patients' blood samples, were directed against α-difluoromethylornithine, parasite numbers significantly reduced in culture, whereas the levels of IFN-γ and IL-12, and the production of reactive oxygen species and nitric oxide, were significantly elevated. Therefore, we demonstrated cross-talk with the use of α-difluoromethylornithine which can reduce the activity of ornithine decarboxylase of L. donovani, eliminating the parasite-induced immune suppression and inducing collateral host protective responses in visceral leishmaniasis.

Different localization of spermidine/spermine N1-acetyltransferase and ornithine decarboxylase transcripts in the rat kidney.

In situ hybridization histochemistry of transverse sections from male rat kidney showed that the mRNA of the regulatory enzyme of polyamine degradation, spermidine/spermine N1-acetyltransferase, has a spotty distribution in the cortex, is low and diffused in the outer stripe and high and diffused in the inner stripe of the outer medulla. At the cellular level, this mRNA is solely expressed by the epithelium of the distal straight and convoluted nephron tubules. Since biosynthetic ornithine decarboxylase mRNA is solely found in the proximal straight tubules, it is proposed that polyamine biosynthesis and degradation occur at separate sites along the nephron.

Analogues of ornithine as inhibitors of ornithine decarboxylase. New deductions concerning the topography of the enzyme's active site.

Fourteen structural analogues of ornithine were synthesized and evaluated as inhibitors of preparations of the enzyme L-ornithine carboxylase (ODC) (E.C. 4.1.1.17) obtained from rat liver, rat hepatoma cells in culture, or bull prostate. The synthesis of these compounds was achieved either via a Bucherer type reaction or via alkylation of carbanions derived from ethyl acetamidocyanoacetate, methyl isocyanoacetate, benzyl alpha-isocyanopropionate, methylbenzaldimine alanate, and the azlactone derivative of ornithuric acid. (+)-alpha-Methylornithine, which was assigned the L configuration on the basis of rotational criteria, was found to be the most potent reversible inhibitor of ODC among the synthesized compounds. From the degree of inhibition of ODC activity in the presence of the various ornithine analogues, it has been possible to delineate some of the structural features of the substrate L-ornithine which are required for binding to the mammalian ODC active site.

Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs.

Ornithine decarboxylase (ODC) from Trichomonas vaginalis was inhibited irreversibly by several substrate analogs. Of these, DL-alpha-monofluoromethyldehydroornithine (MFMDO) and DL-alpha-monofluoromethylornithine (MFMO) were the most potent. The enzyme was unaffected by putrescine analogs suggesting that differences exist between the regulation of the trichomonad enzyme and that in other eukaryotes. In culture the ornithine analogs strongly inhibited putrescine synthesis and increased the generation time after 24 hr of exposure. In a semi-defined growth medium MFMDO methyl and ethyl esters increased the generation time from 4.5 hr to 9.0 and 8.2 hr, respectively. In standard undefined growth medium the trichomonad ODC was fully induced only after 15 hr (late log) and had an extended half-life of greater than 8 hr.

Induction by chloroform of two forms of ornithine decarboxylase in rat liver. Half-life of isozymes.

Ornithine decarboxylase (ODC) in rat liver was separated into two species by DEAE-Sepharose CL-6B column chromatography. The activity of both species of ODC was increased at least 20-fold by chloroform treatment of the rats. The major species, Peak A, contained 65% of the ODC activity and possessed a half-life of 11 min. The second species, Peak B, accounted for 35% of the activity and possessed a half-life of 50 min. The long-lived species of ODC activity, induced in rat liver by chloroform, has not been reported previously and might be related to the prolonged induction of ODC activity by chloroform and to tumor promotion and growth.

Partial purification and characterization of ornithine decarboxylase from Entamoeba histolytica.

Multiplication of E. histolytica was accompanied by a parallel increase in ornithine decarboxylase (ODC) specific activity up to 72 h of cultivation in TYI-S-33 medium. Thereafter, activity rapidly decayed whereas growth continued for another 24 h before entering into the stationary growth phase. ODC was very unstable. Partial purification (14-fold) of the enzyme was achieved by a three-step procedure involving high-speed centrifugation, gel filtration and adsorption to hydroxylapatite. The partially purified enzyme (Mr 211 kDa) revealed maximum activity at pH 8.5-9.0 and a sigmoidal response to substrate concentration. An S0.5 value of 1.0 mM ornithine was estimated. Although ODC did not exhibit an absolute dependence on pyridoxal phosphate (PLP), addition of PLP increased catalytic activity about 4-fold, with an S0.5 value of 45 microM. Evolution of 14CO2 from ornithine was markedly inhibited by polyamines in the following increasing order of effectiveness: putrescine > spermidine > spermine. The substrate analogs alpha-methylornithine and alpha-difluoromethylornithine had no effect on enzyme activity and cell growth. In contrast, 1,3-diaminopropane and 2,4-diamino-2-butanone, 2 putrescine analogs, severely inhibited both enzyme activity and amoeba multiplication. Results are discussed in terms of the role of ODC in the amoeba proliferation.

L-ornithine decarboxylase from Hafnia alvei has a novel L-ornithine oxidase activity.

A novel activity producing gamma-aminobutyric acid (GABA) from L-ornithine in the presence of NAD(P)+ was found in the crude extract of L-ornithine-induced Hafnia alvei, in addition to L-ornithine decarboxylase (ODC) activity. The reaction system for the former activity consisted of two enzymes, L-ornithine oxidase (decarboxylating, OOD) and gamma-aminobutyraldehyde (GABL) dehydrogenase (GDH). OOD catalyzed the conversion of L-ornithine into GABL, CO2, NH3, and H2O2 in the presence of O2, and GDH dehydrogenated GABL to GABA in the presence of NAD(P)+. OOD, purified to homogeneity, had a high ODC activity and the activity ratio of ODC to OOD was almost constant throughout the purification (ODC/ OOD=160:1). The molecular mass of the OOD was about 230 kDa, probably consisting of three identical subunits of a 77 kDa peptide, and OOD had an absorption maximum at 420 nm as well as at 278 nm, the specific absorption for an enzyme containing pyridoxal phosphate (PLP). The content of PLP was estimated at about 1 mol per subunit. OOD was specific to L-ornithine, and other L-amino acids and polyamines including putrescine were inert. The enzyme was activated by PLP, but not by pyridoxamine 5'-phosphate, FAD, FMN, or pyrroloquinoline quinone, and it was inactivated by hydrazine, semicarbazide, and hydroxylamine. The holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted with PLP. These properties of OOD were almost the same as those of ODC separately purified to homogeneity from H. alvei. Zn2+ and Cu2+, butanedione, and sodium borohydride inhibited both OOD and ODC in a similar manner. The OOD reaction required O2 and only the ODC reaction proceeded under anaerobic conditions. The substitution of air for oxygen in the reaction vessel and the addition of catalase-H2O, enhanced only the OOD reaction, resulting in an increase of the ratio of OOD/ODC to 1:30 and 1:4.1, respectively. These results suggested that OOD and ODC are identical and that the former is a side reaction of the latter in the presence of O2.

Purification and some properties of rat intestinal ornithine decarboxylase.

Ornithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr = 53,000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr = 52,000). Values for the Km for L-ornithine (0.1 mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5 x 10(5) nmol CO2/mg protein/30 min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr = 52,000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.

Ornithine decarboxylase as target of chemotherapy.

Ornithine decarboxylase is a key enzyme in polyamine synthesis and growth of mammalian cells. In this chapter I review recent reports on the purification and properties of the pure enzyme, and on the localization, synthesis and regulation of the enzyme in the cell. The use of monospecific antibodies, radiolabeled irreversible inhibitors and cDNA clones for studying enzyme localization, turnover and regulation, is briefly described. This first part is meant to serve as a basis for the analysis of ornithine decarboxylase as a target of chemotherapy. A selection of the most potent inhibitors of ornithine decarboxylase is presented and the effects of some of these in cell culture, in animals and in the clinical setting are reviewed.