Caenorhabditis elegans S-adenosylmethionine decarboxylase is highly stimulated by putrescine but exhibits a low specificity for activator binding

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme of the polyamine synthetic pathway providing decarboxylated S-adenosylmethionine for the formation of spermidine and spermine, respectively. The catalytic activity of the AdoMetDC from the free-living nematode Caenorhabditis elegans highly depends on the presence of an activator molecule. Putrescine, a well-known stimulator of mammalian AdoMetDC activity, enhances the catalytic activity of the nematode enzyme 350-fold. Putrescine stimulation is discussed as a regulatory mechanism to relate putrescine abundance with the synthesis of spermidine and spermine. In contrast to any other known AdoMetDC, spermidine and spermine also represent significant activators of the nematode enzyme. However, the biological significance of the observed stimulation by these higher polyamines is unclear. Although C. elegans AdoMetDC exhibits a low specificity toward activator molecules, the amino acid residues that were shown to be involved in putrescine binding of the human enzyme are conserved in the nematode enzyme. Exchanging these residues by site-directed mutagenesis indicates that at least three residues, Thr192, Glu194 and Glu274, most likely contribute to activator binding in the C. elegans AdoMetDC. Interestingly, the mutant Glu194Gln exhibits a 100-fold enhanced basal activity in the absence of any stimulator, suggesting that this mutant protein mimics the conformational change usually induced by activator molecules. Furthermore, site-directed mutagenesis revealed that at least Glu33, Ser83, Arg91 and Lys95 are involved in posttranslational processing of C. elegans AdoMetDC.     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

Putrescine activation of Trypanosoma cruzi S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that is processed from a single polypeptide into two subunits creating the cofactor. In the human enzyme, both the proenzyme processing reaction and enzyme activity are stimulated by the polyamine putrescine. The processing reaction of Trypanosoma cruzi AdoMetDC was studied in an in vitro translation system. The enzyme was fully processed in the absence of putrescine, and the rate of this reaction was not stimulated by addition of the polyamine. Residues in the putrescine binding site of the human enzyme were evaluated for their role in processing of the T. cruzi enzyme. The E15A, I80K/S178E, D174A, and E256A mutant T. cruzi enzymes were fully processed. In contrast, mutation of R13 to Leu (the equivalent residue in the human enzyme) abolished processing of the T. cruzi enzyme, demonstrating that Arg at position 13 is a major determinant for proenzyme processing in the parasite enzyme. This amino acid change is a key structural difference that is likely to be a factor in the finding that putrescine has no role in processing of the T. cruzi enzyme. In contrast, the activity of T. cruzi AdoMetDC is stimulated by putrescine. Equilibrium sedimentation experiments demonstrated that putrescine does not alter the oligomeric state of the enzyme. The putrescine binding constant for binding to the T. cruzi enzyme (K(d) = 150 microM) was measured by a fluorescence assay and by ultrafiltration with a radiolabeled ligand. The mutant T. cruzi enzyme D174V no longer binds putrescine, and is not activated by the diamine. In contrast, mutation of E15, S178, E256, and I80 had no effect on putrescine binding. The k(cat)/K(m) values for E15A and E256A mutants were stimulated by putrescine to a smaller extent than the wild-type enzyme (2- and 4-fold vs 11-fold, respectively). These data suggest that the putrescine binding site on the T. cruzi enzyme contains only limited elements (D174) in common with the human enzyme and that the diamine plays different roles in the function of the mammalian and parasite enzymes.     

Diethylglyoxal bis(guanylhydrazone), a potent inhibitor of mammalian S-adenosylmethionine decarboxylase. Effects on cell proliferation and polyamine metabolism in L1210 leukemia cells

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1–2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation.     

Mechanisms of allosteric regulation of Trypanosoma cruzi S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.     

Ascaris suum and Onchocerca volvulus: S-adenosylmethionine decarboxylase

Putrescine-dependent S-adenosylmethionine decarboxylase (EC 4.1.1.50) was demonstrated in Ascaris suum and Onchocerca volvulus; activation was found to be about fourfold by putrescine. Mg2+ did not affect the enzyme activity. A. suum was taken as a model nematode and its S-adenosylmethionine decarboxylase was partially purified and characterized. The molecular weight was estimated to be 220,000. The apparent Km-value for adenosylmethionine was determined to be 17 microM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent Ki-values were found to be 0.24 microM and 0.11 microM, respectively. The dependence of filarial worms on uptake and interconversion of putrescine and polyamines as well as properties of the S-adenosylmethionine decarboxylase, different from the host enzyme, points to the polyamine metabolisms as a useful target for chemotherapy.     

Growth and polyamine metabolism inPyrenophora avenae exposed to cyclohexylamine and norspermidine

Summary The effectiveness of inhibitors of polyamine biosynthesis in controlling plant pathogenic fungi is well established. The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated againstin vitro growth of the oat stripe pathogenPyrenophora avenae. Mycelial growth was reduced by 55% upon exposure to 2.0mM CHA while the same concentration of norspermidine reduced growth by 63%. Neither inhibitor had any effect on ODC or AdoMetDC activities, nor the flux of label from ornithine through to the polyamines. Levels of free polyamines in fungal tissue exposed to 0.01 mM norspermidine were unaltered, although 1.0mM CHA did produce a 75% increase in fungal putrescine content. These data suggest that CHA and norspermidine do not reduce fungal growth as a result of a perturbation in polyamine biosynthesis.Abbreviations ODC ornithine decarboxylase - ADC arginine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO adifluoromethylornithine - CHA cyclohexylamine     

Specific regulation by endogenous polyamines of translational initiation of S-adenosylmethionine decarboxylase mRNA in Swiss 3T3 fibroblasts.

S-Adenosylmethionine decarboxylase (AdoMetDC) activity was elevated 18.8-fold in Swiss 3T3 fibroblasts which were depleted of cellular polyamines by using the inhibitor difluoromethylornithine (DFMO). Although the cellular level of AdoMetDC mRNA and the half-life of active AdoMetDC protein were also increased (4.3- and 1.5-fold respectively), together they could not account for the magnitude of the increase in AdoMetDC activity. These data suggested that the translation of AdoMetDC mRNA must be increased in the polyamine-depleted cells to account fully for the elevation in activity. The cellular distribution of AdoMetDC mRNA was examined in the polyamine-depleted cells, and it was found almost exclusively associated with large polysomes. In contrast, AdoMetDC mRNA in untreated controls was very heterogeneous, with the proportion associated with monosomes equal to that associated with large polysomes. The shift of the AdoMetDC message into large polysomes occurred within 18 h after addition of DFMO to the cultures and could be reversed by adding exogenous putrescine. The effect of polyamine depletion on AdoMetDC translation was specific, since there was no change in the distribution in polysomes of either actin mRNA or the translationally controlled mRNA encoding ribosomal protein S16 in the DFMO-inhibited cells. Thus the translational efficiency of AdoMetDC mRNA in vivo is regulated either directly or indirectly by the concentration of intracellular polyamines through a mechanism involving translational initiation, which results in a change in the number of ribosomes associated with this mRNA.     

The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction

In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the C-terminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity approximately 10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Structural biology of S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and a subject of many structural and biochemical investigations for anti-cancer and anti-parasitic therapy. The enzyme undergoes an internal serinolysis reaction as a post-translational modification to generate the active site pyruvoyl group for the decarboxylation process. The crystal structures of AdoMetDC from Homo sapiens, Solanum tuberosum, Thermotoga maritima, and Aquifex aeolicus have been determined. Numerous crystal structures of human AdoMetDC and mutants have provided insights into the mechanism of autoprocessing, putrescine activation, substrate specificity, and inhibitor design to the enzyme. The comparison of the human and potato enzyme with the T. maritima and A. aeolicus enzymes supports the hypothesis that the eukaryotic enzymes evolved by gene duplication and fusion. The residues implicated in processing and activity are structurally conserved in all forms of the enzyme, suggesting a divergent evolution of AdoMetDC.     

Feedback regulation of S-adenosylmethionine decarboxylase synthesis.

Treatment of Ehrlich ascites-tumour cells with 1-amino-oxy-3-aminopropane (AOAP), a potent inhibitor of ornithine decarboxylase, resulted in a marked decrease in cellular contents of putrescine and spermidine, concomitant with an arrest of cell growth. The activity of S-adenosylmethionine decarboxylase (AdoMetDC) was greatly increased in cells treated with AOAP. This increase in AdoMetDC activity was shown to be, at least partly, caused by enhanced synthesis of the enzyme, which most likely was induced by the change in cellular polyamine content.     

Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo

Luminal and basolateral uptake of polyamines by the rat small intestine was studied in vivo. In the concentration range studied (0.1-5 mg per rat) 23-47% of the individual polyamines given intragastrically were found in the body after 1 h, with the small intestine retaining 4-12% of the dose. With spermidine or spermine, labelled polyamines accounted for 85-96% of the counts in the small intestine and between 72-82% were in the form given. However, with putrescine only 29-39% of the label found in the tissue remained in polyamine form and even less, 11-15%, as putrescine. Luminal uptake of polyamines was linear, non-saturable and was not stimulated when small intestinal growth was stimulated by phytohaemagglutinin (PHA). On the basolateral side of the gut, polyamine uptake was stimulated by PHA in a time-dependent way in advance of detectable growth. Overall polyamine recoveries were high (89-99%) with intraperitoneally administered spermidine and spermine. Moreover, a large proportion of the counts in the tissue (63-89%) were still in the original form. Even with putrescine, total recoveries of polyamines (72-88%) and putrescine (24-33%) were elevated in comparison with those from the lumen. Treatment of rats with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, reduced tissue polyamine content, although it had slight effects only on basolateral polyamine transport. The PHA-stimulated increase of polyamine uptake was not abolished in the presence of DFMO.     

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Association of increased polyamine levels with isoproterenol-stimulated mucin secretion in the rat submandibular gland

Incubation of rat submandibular gland slices with 50 microM isoproterenol for 10-40 min stimulated mucin secretion and induced a 3- to 4-fold increase in tissue concentrations of the polyamines putrescine, spermidine and spermine. alpha-Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suppressed the isoproterenol-induced increase in submandibular polyamines and inhibited mucin secretion. Exogenous putrescine restored tissue polyamine levels and partially reversed the inhibitory effect of alpha-difluoromethylornithine on mucin secretion. Rapid increases in polyamine levels appear to mediate isoproterenol-stimulated mucin secretion in the rat submandibular gland.     

Crenarchaeal arginine decarboxylase evolved from an S-adenosylmethionine decarboxylase enzyme

The crenarchaeon Sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. However, genome sequences from S. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5'-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. Instead they have two paralogs of the S-adenosylmethionine decarboxylase (AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both thermostable enzymes self-cleave at conserved serine residues to form amino-terminal beta-domains and carboxyl-terminal alpha-domains with reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine decarboxylation more efficiently than previously studied pyruvoyl enzymes. alpha-Difluoromethylarginine significantly reduced the ArgDC activity of purified enzyme, and treating growing S. solfataricus cells with this inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the beta-subunit of SSO0536 and the alpha-subunit of SSO0585 had ArgDC activity, implicating residues responsible for substrate specificity in the amino-terminal domain. This crenarchaeal ArgDC is the first example of alternative substrate specificity in the AdoMetDC family. ArgDC activity has evolved through convergent evolution at least five times, demonstrating the utility of this enzyme and the plasticity of amino acid decarboxylases.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.