N-methylputrescine Oxidase Research Articles

A class I TGA transcription factor from Tripterygium wilfordii Hook.f. modulates the biosynthesis of secondary metabolites in both native and heterologous hosts

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.

Analysis of the Intracellular Localization of Transiently Expressed and Fluorescently Labeled Copper-Containing Amine Oxidases, Diamine Oxidase and N-Methylputrescine Oxidase in Tobacco, Using an Agrobacterium Infiltration Protocol.

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.

Molecular Evolution of N-Methylputrescine Oxidase in Tobacco

Biosynthesis of nicotine in tobacco requires N-methylputrescine oxidase (MPO), which belongs to the copper-containing amine oxidase superfamily. Previous studies identified tobacco MPO1 and its close homolog NtDAO1 (formerly called MPO2), of which MPO1 has been shown preferentially to oxidize N-methylated amines. We show here that NtDAO1, as well as a homologous Arabidopsis diamine oxidase (DAO), accept non-N-methylated amines more efficiently than their corresponding N-methylated amines. MPO1 is coordinately regulated with other nicotine biosynthesis genes with regard to COI1-MYC2-dependent jasmonate induction and its dependence on nicotine-specific ERF transcription factors, whereas NtDAO1 is constitutively expressed at low basal levels in tobacco plants. Both MPO1 and NtDAO1 are targeted to peroxisomes by their C-terminal motifs, and the peroxisomal localization of MPO1 is required for it to function in nicotine biosynthesis in jasmonate-elicited cultured tobacco cells. Restricted occurrence of the MPO subfamily in Nicotiana and Solanum indicates that, during the formation of the Solanaceae, MPO has evolved from a DAO, which functions in polyamine catabolism within peroxisomes, by optimizing substrate preference and gene expression patterns to be suitable for alkaloid formation.

Why does Anatabine, But not Nicotine, Accumulate in Jasmonate-Elicited Cultured Tobacco BY-2 Cells?

Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

Molecular Cloning of N-methylputrescine Oxidase from Tobacco

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida× aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.

Purification of N-methylputrescine oxidase from Nicotiana rustica

The yield of N-methylputrescine oxidase extracted from Nicotiana rustica roots grown in organ culture was improved by supplementing White's medium with 1.15 mM (NH 4) 2SO 4. The enzyme was purified from these roots by salt fractionation, anion exchange, affinity and hydrophobic interaction chromatography. The purified protein had M r 54 000 by SDS-PAGE.

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of α-naphthalene-acetic acid, kinetin (N(6)-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.

Quinoprotein characteristics of N-methylputrescine oxidase from tobacco roots

N-Methylputrescine oxidase (MPO) was partially purified from Nicotiana tabacum roots by ammonium sulphate fractionation, anion-exchange chromatography, and metal chelate chromatography. Analytical-scale gel filtration HPLC in the presence of detergent reduced the composition of the final preparation to six protein species. Several amine oxidase inhibitors were screened for inhibition of partially-purified MPO. Phenylhydrazine and dinitrophenylhydrazine were the most inhibitory. Inactivation by phenylhydrazine had characteristics typical of ‘suicide’ inhibition, suggesting that MPO is a quinoprotein. Treatment of partially-purified MPO with [ 14C]-labelled phenylhydrazine yielded a radiolabelled band of ca 70 000 M r on SDS-PAGE.

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ 1-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ 1-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.

Nicotine Biosynthetic Enzyme Activities in Nicotiana tabacum L. Genotypes with Different Alkaloid Levels

Young plants of five Nicotiana tabacum L. genotypes were examined for activity of nicotine biosynthetic enzymes. Genotypes near isogenic except at two loci each with two alleles controlling nicotine level were used in a comparison of the four homozygous allelic combinations producing high, high intermediate, low intermediate, and low nicotine levels in a "Burley 21" background. Putrescine N-methyltransferase (EC 2.1.1.53) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) activities in root tissue of these four genotypes were proportional to leaf nicotine level, whereas N-methylputrescine oxidase activity in root tissue differed in proportion and ranking. Quinolinic acid phosphoribosyltransferase activities in leaf tissue were lower than in roots, but no differences were found among the four genotypes. The homozygous recessive alleles at either locus affect levels of all three enzyme activities examined in roots. Each locus seems to be involved in regulation of nicotine metabolism, but whether directly as a regulatory locus or indirectly through the metabolic product of a structural locus is not known.No difference was observed between enzymic oxidation of putrescine and N-methylputrescine by leaf and root extracts of Burley 21 (a high nicotine, low nornicotine genotype) and a high nornicotine cultivar, "Robinson Medium Broadleaf." Putrescine was utilized as a substrate to a greater extent than N-methylputrescine by leaf extracts compared with root extracts of both cultivars. It was concluded that genetic differences in levels of nicotine and nornicotine were not due to differences in enzymic oxidation of these two precursors during alkaloid biosynthesis.

Changes in the activities of ornithine decarboxylase, putrescine N-methyltransferase and N-methylputrescine oxidase in tobacco roots in relation to nicotine biosynthesis<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Changes in ornithine decarboxylase, putrescine N-methyltransferase and N-methylputrescine oxidase activities in response to some treatments were investigated using hydroponically grown tobacco plants. Decapitation of shoots brought about marked elevation of the three enzyme activities, which reached their peaks 24 hr after decapitation, then declined. An excellent correlation was observed between the accumulation of nicotine and changes in the three root enzyme activities. Administration of IAA at 2.5 to 5 μconcentration significandy increased these enzyme activities in roots of decapitated plants but higher concentrations of IAA prevented the rise in enzyme activities promoted by decapitation. Nicotine strongly inhibited the rise in enzyme activities in roots of decapitated plants in all cases. The results suggest that these enzymes are under the control of a common regulatory system, in which auxin and nicotine are important components. Ornithine decarboxylase was present in all the plants examined, but putrescine N-methyltransferase and N-mediylputrescine oxidase were detected only in the roots of tobacco, Datura and Atropa plants.

N-methylputrescine oxidase from tobacco roots

N-Methylputrescine oxidase, catalyzing the oxidative deamination of the primary amino group of N-methylputrescine, was demonstrated in roots of tobacco plants, and purified 150-fold. N-Methylpyrrolinium salt was identified as the reaction product by comparison with the authentic compound. The enzyme had a pH optimum at 8·0 and the K m value for N-methylputrescine was 4·5 × 10 −4 M. Putrescine and cadaverine were also oxidized. Activity of the enzyme was strongly inhibited by carbonyl reagents, thiol inhibitors and diethyldithiocarbamate, but hydrazine derivatives were without significant effect on the enzyme activity. The enzyme was localized exclusively in the roots and its activity markedly increased on decapitation of the shoots. The demonstration of N-methylputrescine oxidase, in addition to putrescine N-methyltransferase, provides evidence that the biosynthetic route of nicotine involves N-methylputrescine and 4-methylaminobutanal ( N-methylpyrrolinium salt) as intermediates.