Spermidine Synthase Research Articles

Comparative protein modeling of spermidine synthase from Plasmodium falciparum: A potential target for anti-malarial drug therapy

Malaria, caused by protozoan parasites of the genus Plasmodium, affects up to 500 million individuals and kills over 1 million people every year. The increasing resistance of the malaria parasites has enforced strategies for finding new drug targets. In recent years, enzymes associated with the polyamine metabolism have attracted attention as drug targets. Cytosolic Plasmodium falciparum spermidine synthase (PfPAPT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite polyamine amine biosynthesis, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper I propose a theoretical low-resolution 3D model for PfPAPT based on crystal structure of the Arabidopsis thaliana, by multiple alignment followed by intensive optimization; validation and dynamic simulations in water. Comparison between the active sites of PfPAPT and human PAPT revealed key differences that could be useful for the design of new selective inhibitors of Plasmodium PAPT.

Differential distribution of transcripts from genes involved in polyamine biosynthesis in bean plants

Partial cDNAs sequences for arginine decarboxylase (Pvadc), S-adenosylmethionine decarboxylase (Pvsamdc) and spermidine synthase (Pvspds) were isolated from the bean Phaseolus vulgaris using primers designed from conserved regions of enzymes belonging to plant species. Sequence analysis showed that the Pvadc, Pvsamdc and Pvspds genes were most closely related to the orthologous genes from Glycine max, Phaseolus lunatus and Pisum sativum, respectively. The expression patterns of the genes, together with that of ornithine decarboxylase (Pvodc), were analysed in young and mature leaves, stems, roots, root tips, petals, stigma, ovaries, filaments and anthers of bean plants. Pvsamdc was found to be expressed at similar levels in all tissues. The other transcripts showed tissue specific expression. Pvadc was barely expressed in petals and not at all in roots tips, Pvspds was mainly expressed in roots, stigma and filaments, and Pvodc was detected only in roots.

Leishmania donovani Polyamine Biosynthetic Enzyme Overproducers as Tools To Investigate the Mode of Action of Cytotoxic Polyamine Analogs

A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.

Abscisic acid modulates polyamine metabolism under water stress in Arabidopsis thaliana

Limited information is available concerning plant signal transduction pathways regulating polyamine (PA) metabolism in response to various abiotic stress conditions. To gain a new insight into this question, we have studied the possible role of abscisic acid (ABA) in the modulation of PA metabolism under water stress conditions in the model plant Arabidopsis thaliana. Transcriptome profiling of the PA‐biosynthetic gene family by real‐time quantitative reverse transcriptase polymerase chain reaction revealed that arginine decarboxylase 2 (ADC2), spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) genes are highly induced by drought stress, and these responses are mostly impaired in ABA‐deficient (aba2‐3) and ABA‐insensitive (abi1‐1) mutants. These data, together with PA content analyses, indicate that ABA modulates PA metabolism at transcriptional and metabolite level in response to water stress.

Assay for spermidine synthase activity by micellar electrokinetic chromatography with laser-induced fluorescence detection

An assay for spermidine synthase (SPDS) activity in rat liver has been developed using micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection to enable the discovery of SPDS inhibitors. The assay was established by estimating the amount of spermidine (SPD) produced from the putrescine (PUT) present by SPDS. The SPD in an enzyme reaction mixture of homogenized rat liver could directly react with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent. The NBD derivatives of SPD and PUT could be separated and detected by MEKC-LIF detection within 15 min. The IC 50 value measured for SPDS inhibitor, 4-methylcyclohexylamine, in rat liver by this assay was consistent with published data. Our SPDS assay using MEKC-LIF is simple and allows easy determination of SPDS activity in homogenized samples without troublesome procedures such as preparation of antibody or fluorescence-labeled substrate. The assay should be effective for discovering the SPDS inhibitors using biological samples.

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.

Regulation of Polyamine and Cancer

This review describes my work in the field of polyamine research for the last 35 years. My research started with developing the improved synthesis of decarboxylated S-adenosylmethionine and then moved to the purification of spermidine synthase from rat prostate. I also took considerable efforts to find the synthetic procedure for various polyamines with high yield in order to prepare (15)N-labeled polyamines. On the basis of these methodological work, I searched for the inhibitor of spermidine synthase and found trans-4-methylcyclohexylamine (MCHA), the most effective one at the present time. I also developed a new analytical method for polyamines using stable isotope and ionspray ionization mass spectrometry (IS-MS). Based on these studies I examined the role of polyamines in liver regeneration and found that oral administration of MCHA effectively changed the concentration of polyamines and inhibited the hepatic growth. I also found the close relationship between the concentration ratio of spermidine to spermine and the extent of liver regeneration. These results may shed new light on the control of cell growth by polyamine in vivo.

Role of polyamines in peach fruit development and storage

To investigate the role of polyamines in pre- and post-harvest fruit development of 'Akatsuki' peach (Prunus persica (L.) Batsch.) we measured polyamine concentrations, activities of polyamine biosynthetic enzymes and expression of genes encoding these enzymes. Concentrations of the free polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) in pre-harvest fruit peaked 16 days after full bloom (DAF) and then progressively decreased until harvest with the exception of Put, which showed a second peak at 94 DAF, just before the onset of ethylene production. In post-harvest fruit, minor changes in concentrations of Spd and Spm were observed, whereas Put concentration peaked on the harvest day, followed by an abrupt decrease and a subsequent 2-fold increase, which was opposite to the fluctuating pattern of ethylene production. Activities of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) peaked during the first stage of fruit development and then decreased until 80 DAF, after which the activities were below detection limits, suggesting that Put is synthesized during the early stage of fruit development. Activity of S-adenosylmethionine decarboxylase (SAMDC) decreased progressively until the end of S2. Expression levels of five putative polyamine biosynthetic genes, ADC, ODC, SAMDC, spermidine synthase (SPDS) and spermine synthase (SPMS), in pre-harvest and post-harvest fruit did not coincide precisely with the observed changes in enzymatic activities and polyamine concentrations. The possible role of polyamines during peach fruit development and the relationship between polyamines and ethylene biosynthesis are discussed.

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1Δ mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase ( meu1Δ). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1Δ cells. By comparing the results with this mutant with a meu1Δ spe2Δ mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1 + SPE2 + cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15Δ mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

AbstractIn order to determine whether hydrogen peroxide (H2O2) generated by polyamine oxidative degradation is involved in the development of lateral roots in soybean, the length and the number of lateral roots, the activities of polyamine oxidases and diamine oxidases, and the endogenous free polyamine and H2O2 content were analyzed in soybean (Glycine max (Linn.) Merr.) main roots of 2‐d‐old seedlings after treatments for 2 d with exogenous β‐hydroxyethylhydrazine (an inhibitor of polyamine oxidases), H2O2, putrescine, cyclohexylamine (an inhibitor of spermidine synthase) or N, N′‐dimethylthiourea (a scavenger of hydrogen peroxide). β‐hydroxyethylhydrazine treatment strongly inhibited the development of lateral roots in soybean seedlings, reduced the activities of polyamine oxidases and diamine oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamines in the main roots. The inhibitory effect of β‐hydroxyethylhydrazine on root development could be alleviated by exogenously applied 10 μmol/L H2O2 (a major product of polyamine oxidation). Treatment with cyclohexylamine and putrescine promoted root growth slightly, but treatment with cyclohexylamine plus N, N′‐dimethylthiourea or putrescine plus N, N′‐dimethylthiourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes in endogenous H2O2 levels. These results suggest that the development of soybean lateral roots is associated with the oxidative degradation of polyamines, and that their products, especially H2O2, are likely to play an important role in the growth of soybean lateral roots.(Managing editor: Ping He)

Annealing control primer system identifies differentially expressed genes in blastocyst-stage porcine parthenotes

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.

Effect of Salt Stress on the Regulation of Maize (Zea mays L.) Genes Involved in Polyamine Biosynthesis

A cDNA for spermidine synthase (SPDS), which converts putrescine to the higher polyamine spermidine using decarboxylated S-adenosylmethionine as a cofactor, was isolated from Zea mays leaves (Zmspds2A). Comparison of the deduced amino acid sequence revealed a high homology (81.9%) with Oryza sativa SPDS2. RT-PCR analyses showed that Zmspds2A was equally expressed in leaves, stem and roots. In contrast, transcripts of other genes related to polyamine biosynthesis (Zmodc, adc and samdc) showed tissue-specific regulation. The effect of salt stress on the expression of all these genes in maize leaves exposed to NaCl solutions of different concentrations was analysed. Our results showed that only Zmodc and Zmspds2A were up-regulated by salt stress; whereas the other two genes were barely affected by this treatment. In addition to Zmspds2A, a second transcript encoding a maize spermidine synthase (Zmspds2B) that also became up-regulated by salt stress, was identified. Comparison of partial cDNA sequences of transcripts Zmspds2A and Zmspds2B with the corresponding genomic DNA region revealed the existence of alternative splicing mechanism, opening a new aspect in plant polyamine biosynthesis modulation under abiotic stress.

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence

Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.

Aminopropyltransferases: Function, Structure and Genetics

Aminopropyltransferases use decarboxylated S-adenosylmethionine as an aminopropyl donor and an amine acceptor to form polyamines. This review covers their structure, mechanism of action, inhibition, regulation and function. The best known aminopropyltransferases are spermidine synthase and spermine synthase but other members of this family including an N(1)-aminopropylagmatine synthase have been characterized. Spermidine synthase is an essential gene in eukaryotes and is very widely distributed. Key regions in the active site, which are very highly conserved, were identified by structural studies with spermidine synthase from Thermotoga maritima bound to S-adenosyl-1,8-diamino-3-thiooctane, a multisubstrate analog inhibitor. A general mechanism for catalysis by aminopropyltransferases can be proposed based on these studies. Spermine synthase is less widely distributed and is not essential for growth in yeast. However, Gy mice lacking spermine synthase have multiple symptoms including a profound growth retardation, sterility, deafness, neurological abnormalities and a propensity to sudden death, which can all be prevented by transgenic expression of spermine synthase. A large reduction in spermine synthase in human males due to a splice site variant causes Snyder-Robinson syndrome with mental retardation, hypotonia and skeletal abnormalities.

Improvement of environmental stress tolerance of sweet potato by introduction of genes for spermidine synthase

With the purpose of enhancing environmental stress tolerance of sweet potato (Ipomoea batatas, cv. Kokei 14), we transformed this plant with spermidine synthase genes derived from Cucurbita ficifolia (FSPD1). The FSPD1-transgenic plants showed twice as high spermidine content as the wild type (WT) counterpart in both leaves and storage roots. One of the most characteristic features of the transgenic plants was the increase in the number of storage roots formed under both non-stress and stressful environments. Salt and drought stresses suppressed storage root growth, but the transgenic plants were less affected producing significantly larger mass of storage roots and starches than WT plants under either stress. The transgenic plants also showed increased tolerance to chilling- and heat-mediated damage to photosynthesis compared to the WT plants. Thus, sweet potato was made more tolerant to environmental stresses through introduction of the FSPD1 genes. This improved tolerance may involve enhanced oxidative stress tolerance at least partially because the transgenic plants were more tolerant to paraquat, a potent oxidative stress inducer, than the WT plants. From these results, the FSPD1 gene is considered useful for gene transfer technology aiming at improving environmental stress tolerance of sweet potato.

Studies on the regulation of ornithine decarboxylase in yeast: Effect of deletion in the MEU1 gene

Methylthioadenosine is formed during the biosynthesis of spermidine and of spermine and is metabolized by methylthioadenosine phosphorylase, an enzyme missing in several tumor cell lines. In Saccharomyces cerevisiae, this enzyme is coded by the MEU1 gene. We have now studied the effect of the meu1 deletion on polyamine metabolism in yeast. We found that the effects of the meu1Delta mutation mostly depend on the stage of cell growth. As the cell density increases, there is a marked fall in the level of ornithine decarboxylase (ODC) in the MEU1(+) cells, which we show is caused by an antizyme-requiring degradation system. In contrast, there is only a small decrease in the ODC level in the meu1Delta cells. The meu1Delta cells have a higher putrescine and a lower spermidine level than MEU1(+) cells, suggesting that the decreased spermidine level in the meu1Delta cultures is responsible for the greater apparent stability of ODC in the meu1Delta cells. The lower spermidine level in the meu1Delta cells probably results from an inhibition of spermidine synthase by the methylthioadenosine that presumably accumulates in these mutants. In both MEU1(+) and the meu1Delta cultures, the ODC levels were markedly decreased by the addition of spermidine to the media, and thus our results contradict the postulation of Subhi et al. [Subhi, A. L., et al. (2003) J. Biol. Chem. 278, 49868-49873] of a novel regulatory pathway in meu1Delta cells in which ODC is not responsive to spermidine.

Cloning, expression, characterisation and three-dimensional structure determination of Caenorhabditis elegans spermidine synthase

The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S-adenosylmethionine ( K m = 110 μM) and a less pronounced feedback inhibition by the second reaction product 5’-methylthioadenosine (IC 50 = 430 μM). The C. elegans protein that carries a nematode-specific insertion of 27 amino acids close to its N-terminus was crystallized, leading to the first X-ray structure of a dimeric eukaryotic SPDS.

Two types of spermine synthase gene: MdACL5 and MdSPMS are differentially involved in apple fruit development and cell growth

Three cDNAs with high homology to spermine (Spm) synthases in Arabidopsis were isolated from apple [ Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. MdACL5-1 and MdACL5-2 have high homology with ACL5 and MdSPMS has high homology with AtSPMS. The similarity of MdSPMS to spermidine synthases (SPDSs) was higher than that of MdACL5s, despite the fact that both are putative Spm synthases. However, MdSPMS could be discriminated from SPDSs by the presence of several characteristic amino acids, i.e., Val-149, Ser-161, Ala-205, and Val-235, in the decarboxylated S-adenosylmethionine (dcSAM)-binding motif of MdSPMS. Both MdACL5-1 and MdSPMS complemented Spm biosynthesis in a yeast mutant deficient in Spm synthase, and ectopic expression of MdACL5-1 in the Arabidopsis dwarf mutant acl5 allowed recovery of the normal phenotype. RNA gel blot analysis showed that MdACL5 and MdSPMS are differentially expressed in tissues and suspension cells. These results suggest that functional MdACL5 and MdSPMS are independently involved in apple fruit development and cell growth.

Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant

Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the first specific step in the biosynthesis of tropane and nicotine alkaloids. Potato (Solanum tuberosum L.) contains neither nicotine nor the medicinal tropane alkaloids hyoscyamine or scopolamine, but calystegines. They are nortropane alkaloids with glycosidase inhibitory activity. Based on the assumption of calystegine formation by the tropane alkaloid pathway, PMT genes and enzymes were investigated in potato. Sprouting tubers contained both N-methylputrescine and PMT activity. Two cDNA clones coding for PMTs were obtained together with a cDNA clone for spermidine synthase (SPDS, EC 2.5.1.16). The pmt sequences resemble those from Nicotiana tabacum (85% identity) and those from tropane alkaloid plants, Atropa belladonna (80% identity) and Hyoscyamus niger (79% identity). They are less similar to SPDS of S. tuberosum (66% identity). Expression of pmt1 and spds cDNA in Escherichia coli yielded active enzymes, while pmt2 expression resulted in insoluble protein. Chimera proteins obtained by fusion of fragments of S. tuberosum pmt2 and H. niger pmt were active as PMT, if the initial part of pmt2 was used, indicating that a mutation in the terminal part of the gene caused insolubility of the enzyme. PMT1 was purified after expression in E. coli and proved to be an active N-methyltransferase without SPDS activity. The enzyme was specific for putrescine (K (M) 250 microM) and inhibited by n-butylamine and cadaverine. While spds was transcribed in all plant organs, pmt transcripts were found in small tuber sprouts only. The results confirm that in potato genes and enzymes specific for the tropane alkaloid metabolism are expressed and active.