Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity

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.     

Arginine decarboxylase as the source of putrescine for tobacco alkaloids

The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.     

Endogenous polyamine levels in macrophages is sufficient to support growth of Toxoplasma gondii

Cytotoxic-activated macrophages control Toxoplasma gondii growth by producing nitric oxide (NO). However, the parasite can partially inhibit NO production. NO is generated from arginine within the polyamine biosynthetic pathway. Two enzymes of this pathway are ornithine, decarboxylase (ODC) and arginine decarboxylase (ADC). The aim of the present work was to investigate whether T. gondii is able to modulate polyamine metabolism in macrophages. Toxoplasma gondii infection did not affect basal ODC or ADC activity. However, lipopolysaccharide induced an increase in ODC activity. Polyamine-treated macrophages exhibited a T. gondii-infection index similar to controls but a higher adhesion index; the parasite did not grow in methyl-ornithine (ODC inhibitor)-treated macrophages. The parasites were able to take up putrescine with a Km of 0.92 microM, indicating the presence of a high-affinity putrescine-transporter system. Putrescine-treated T. gondii actively penetrated macrophages and Vero cells. However, NO production and lysosomal parasitophorous vacuole fusion were not inhibited. Considered together, these results demonstrate that T. gondii requires polyamines for multiplication. However, as opposed to Trypanosoma cruzi and because of a relatively high-affinity putrescine-transporter system in the parasite, constitutive macrophage levels of putrescine seem sufficient to support T. gondii survival and multiplication.     

Participation of ornithine decarboxylase in early stages of tomato fruit development

The apparent association of ornithine decarboxylase (ODC) with rapid cell proliferation in developing tomato (Lycopersicon esculentum Mill. cv. Pearson ms-35) fruits has been previously described. Further evidence is provided by the use of two ODC inhibitors, α-difluoromethylornithine (α-DFMO) and α-methylornithine (α-MO). Fruit development was inhibited by these inhibitors if applied during the period of intensive cell division. When applied in vitro, the two inhibitors were shown to inhibit the activity of ODC but not that of arginine decarboxylase (ADC). When applied in vivo, α-DFMO, a catalytic irreversible inhibitor, caused 97.1% reduction of ODC activity in the dialyzed extract from the treated ovaries, while it had no effect on ADC. On the other hand, α-MO, a reversible inhibitor, did not reduce the activity of these two enzymes in the dialyzed extracts when applied in vivo. The dialysis procedure probably removed α-MO from the enzyme fraction. Putrescine, the product of both ODC and ADC, alleviated the inhibition of fruit development but did not restore ODC activity to the control level. These results suggest that in the young developing tomato fruit, ODC is the enzyme responsible for the synthesis of putrescine, which is essential for the early stages of fruit development. The reduced activity of ODC elicited by putrescine suggests a mechanism of feedback regulation by enzyme repression or release of an ODC anti-enzyme.     

Arginine decarboxylase as the source of putrescine for tobacco alkaloids

The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source ofputrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation ofputrescine going into alkaloids: (a) A specific ‘suicide inhibitor’ of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of ¹⁴C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.     

Activities and properties of putrescine-biosynthetic enzymes in Vibrio parahaemolyticus

The biosynthetic pathways for putrescine (Put) in Vibrio parahaemolyticus were delineated by measuring activities of the enzymes which would be involved in its biosynthesis. Experiments with labeled arginine and ornithine revealed that both of these amino acids were converted into Put by intact cells. The activities of three enzymes, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and agmatine ureohydrolase (AUH), were detected in cell extracts. ADC and ODC of V. parahaemolyticus were similar in the following properties to the corresponding enzymes of Escherichia coli: 1) both decarboxylases showed a pH optimum at 8.25 and required pyridoxal phosphate and dithiothreitol for full activity; 2) while ODC was considerably activated by GTP, ADC was only slightly; 3) both decarboxylases were inhibited by polyamines; 4) ADC was inhibited by difluoromethylarginine, a potent inhibitor of bacterial ADC. However, in contrast to the corresponding enzymes of E. coli, the V. parahaemolyticus ADC showed no requirement for Mg2+, and the AUH was active over a wide pH range of 8.5-9.5 with a maximum at pH 9.0. Furthermore, in all 6 strains tested, the activity of ADC was obviously high compared with that of ODC, and AUH was present with a relatively high activity. Cultivation of these strains at a suboptimal NaCl concentration (0.5%) resulted in a pronounced increase in both ADC and AUH activities. These observations suggest that the important pathway for Put biosynthesis in V. parahaemolyticus is the decarboxylation of arginine by ADC and the subsequent hydrolysis of its product, agmatine, by AUH.     

DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [( 3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.     

Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli.

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.     

Biosynthetic arginine decarboxylase in phytopathogenic fungi

It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Lack of arginine decarboxylase in Trypanosoma cruzi epimastigotes

The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild-type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor omithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild-type T. cruzi epimastigotes lack ADC enzymatic activity.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Polyamine biosynthesis in Phytomonas: Biochemical characterisation of a very unstable ornithine decarboxylase

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5′-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.     

Polyamine metabolism,floral initiation and floral development in Chrysanthemum (Chrysanthemum morifolium Ramat.)

In the short-day plant Chrysanthemum (Chrysanthemum morifolium Ramat. variety Pavo) putrescine and spermidine conjugates appeared in the apical bud before the first observable transformation of the meristem into floral structures. These compounds accumulated on floral initiation and well before floral evocation. Spermidine conjugates were predominant during floral initiation whereas free amines did not accumulate to any significant extent. Different associations of amides were observed during floral initiation as compared with the reproductive phase. 3,4-Dimethoxyphenethylamine conjugates (water-insoluble compounds) were the predominant amine conjugates observed during flower development. These compounds decreased drastically after fertilization. In vegetative buds from plants grown in long days polyamine conjugates were very low and appeared as plants aged. We present evidence that ornithine decarboxylase (ODC) regulates putrescine biosynthesis during floral initiation and floral development. When ODC action was blocked by DFMO (-DL-difluoromethylornithine, a specific, irreversible inhibitor of ODC), flowering was inhibited, and free and conjugated polyamines were not detected. This treatment led to a slight enhancement of ADC activity. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with DFMA (-DL difluoromethylarginine, a specific, irreversible inhibitor of ADC) did not affect flowering and the polyamine titers. The results suggest that ODC and polyamine conjugates are involved in regulating floral initiation in Chrysanthemum.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine     

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.     

The possible involvement of polyamines in the development of tomato fruits in vitro

The apparent involvement of ornithine decarboxylase (ODC) and putrescine in the early stages of fruit growth in tomato (Lycopersicon esculentum Mill.) has been previously described. Further evidence presented here supports the direct involvement of ODC and putrescine in the cell division process in tomato fruits. In tomato fruits grown in vitro, in which basic growth processes are inhibited, the activity of ODC and arginine decarboxylase (ADC) and the level of free polyamines were reduced. While ODC and ADC activity was correlated with the period of cell division in the tomato fruit, the free polyamine content was correlated with the DNA content, cell size, and fruit fresh weight. The addition of exogenous putrescine, however, did not restore the basic growth processes in the fruits grown in vitro.