Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG polyethylene glycol - PMT putrescine-N-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support.     

Changes in the activities of ornithine decarboxylase, putrescine N-methyltransferase and N-methylputrescine oxidase in tobacco roots in relation to nicotine biosynthesis

Changes in ornithine decarboxylase, putrescine N-methyltransferaseand N-methylputrescine oxidase activities in response to sometreatments were investigated using hydroponically grown tobaccoplants. Decapitation of shoots brought about marked elevationof the three enzyme activities, which reached their peaks 24hr after decapitation, then declined. An excellent correlationwas observed between the accumulation of nicotine and changesin the three root enzyme activities. Administration of IAA at2.5 to 5 µconcentration significandy increased these enzymeactivities in roots of decapitated plants but higher concentrationsof IAA prevented the rise in enzyme activities promoted by decapitation.Nicotine strongly inhibited the rise in enzyme activities inroots of decapitated plants in all cases. The results suggestthat these enzymes are under the control of a common regulatorysystem, in which auxin and nicotine are important components.Ornithine decarboxylase was present in all the plants examined,but putrescine N-methyltransferase and N-mediylputrescine oxidasewere detected only in the roots of tobacco, Datura and Atropaplants. ¹Part XVI of the series "Phytochemical Studies on Tobacco Alkaloids". (Received August 1, 1972; )     

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.     

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.     

Phytochemical and cytotoxic studies on the roots of Euphorbia fischeriana

With the aim of supporting the folk applications of Euphorbia fischeriana, a phytochemical study was performed, which led to the discovery of 9 compounds, including three new ones (1–3) and six known ones (4–9). Their structures were determined by 1D, 2D NMR, and HRESIMS analysis. In the cytotoxic assays on Hep-3B cell line, 2 showed stronger inhibitory effects (IC₅₀ 8.1 μmol/L) than that of positive control, and 1, 8 and 9 also gave inhibitory effects in a certain degree with IC₅₀ values of 12.5, 12.0 and 18.7 μmol/L, respectively. While on A549, the cytotoxic activities of 1 (IC₅₀ 11.9 μmol/L) and 8 (IC₅₀ 9.4 μmol/L) were superior to that of 5-Fu, and those of 4 and 9 were moderate with IC₅₀ values of 28.2 and 29.8 μmol/L, respectively. In addition, both petroleum ether and dichloromethane extracts showed cytotoxic activities with different degree, while n-butanol extracts had no effect. The results clarified that the low-polarity fractions of E. fischeriana, including triterpenoids, abietane and tigliane-type diterpenoids might be the potential bioactive ingredients which will exert strong antitumor effects.     

Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle.

The cDNAs encoding putrescine N-methyltransferase (PMT), which catalyzes the S-adenosylmethionine-dependent N-methylation of putrescine at the first committed step in the biosynthetic pathways of tropane alkaloids, were isolated from Atropa belladonna and Hyoscyamus niger. These PMTs, however, lacked the N-terminal tandem repeat arrays previously found in Nicotiana PMTs. AbPMT1 RNA was much more abundant in the root of A. belladonna than was AbPMT2 RNA. The 5'-flanking region of the AbPMT1 gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to A. belladonna. Histochemical analysis showed that GUS is expressed specifically in root pericycle cells and that the 0.3-kb 5'-upstream region was sufficient for pericycle-specific expression. Treatment of A. belladonna roots with methyl jasmonate did not up-regulate the expression of GUS or endogenous AbPMT genes. The regulation of tropane alkaloid biosynthesis is discussed and compared with that of nicotine biosynthesis.     

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.     

Effect of agmatine on putrescine formation in tobacco plants

Incorporation of L-arginine-U-₁₄C, fed to leaf disks of tobaccoplants, into putrescine decreased about 70% in the presenceof agmatine, but that of L-ornithine-U-₁₄C decreased only slightly.These results indicate that putrescine is synthesized from argininevia agmatine, but is also synthesized from ornithine withoutpassing through arginine and agmatine. (Received April 23, 1969; )     

Purification and properties of pancreatic glycine N-methyltransferase.

Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.     

Theoretical studies on the conformation of saccharides. XIV. Structure and conformational properties of the glycosylamines

The 2-methylaminotetrahydropyran was used as a model to study conformational properties of the N-glycosidic linkage in glycosylamines. Relaxed two-dimensional conformational (phi, psi) maps in 20 solvents were calculated by a method in which the total energy is divided into the energy of the isolated molecule and the solvation energy. Molecular geometry optimization has been carried out for each conformer using the quantum chemical method PCILO. The calculated variations of the geometry are consistent with the results obtained by the statistical analysis of available experimental data retrieved from the Cambridge Structural Database. The calculated abundances of conformers show that the polarity of the solvent has little effect on the anomeric ratio, and the form having the methylamino group equatorial is favored in all considered solvents.     

Phytochemical and chemotaxonomic studies on Pteris wallichiana J. Agardh

A systematic phytochemical investigation of Pteris wallichiana J. Agardh resulted in the isolation of twenty compounds, including five sesquiterpenes (1–5), six flavonoids (6–11), seven phenolic acids (12–18) and two fatty acids (19 and 20). Their structures were deduced from MS, NMR and ORD data. This is the first report of compounds dehydropterosin B (2), (2R,3S)-pterosin C (4), (2R,3R)-pterosin L (5), apigenin (6), luteolin (7), luteolin-7-O-glucoside (10), caffeic acid (13), vanillin (14), 3,4-dihydroxybenzaldehyde (15), chlorogenic acid (17), 3,5-dicaffeoylquinic acid (18), suberic acid (19) and azelaic acid (20) from P. wallichiana and of compounds 15, 19 and 20 from the family Pteridaceae. Furthermore, a chemotaxonomic study of the isolates was performed.     

Formation of putrescine from ornithine and arginine in tobacco plants

Putrescine was formed when both DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴Cwere injected into tobacco plants. The incorporation rate ofthese amino acids into putrescine was higher in potassium orsulfur deficient plants than in normal plants. Degradation ofputrescine-1,4-¹⁴C injected into tobacco plants was inhibitedby a potassium or sulfur deficiency. Increased accumulationof putrescine in potassium or sulfur deficient plants may bepartly dependent upon this inhibition in the degradation ofputrescine. Detached leaves and roots were also able to formputrescine from DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴C. (Received December 13, 1968; )     

Formation of putrescine and cinnamoyl putrescines in tobacco cell cultures

A p-fluorophenylalanine- (PFP) resistant cell line of Nicotiana tabacum and wild type cells accumulating high and low levels of cinnamoyl putrescines, respectively, were used to study the formation of putrescine in the biosynthesis of cinnamoyl putrescines. Labelled arginine and ornithine were equally well incorporated into the main conjugates caffeoyl and feruloyl putrescine. Trapping experiments indicated that both amino acids were decarboxylated for putrescine biosynthesis. Nearly all alcohol-extractable radioactivity from the labelled amino acids was found as cinnamoyl putrescines in the PFP-resistant cell line, whereas wild type cells retained significant radioactivity in the amino acids. The enzyme activities of arginine and ornithine decarboxylases in the resistant cell line were increased 3- to 6-fold.     

Functional analysis of the Rubisco large subunit ÂN-methyltransferase promoter from tobacco and its regulation by light in soybean hairy roots

The ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) large subunit (LS) ^ɛ N-methyltransferase (Rubisco LSMT) catalyzes post-translational methylation of the ɛ-amino group of lysine-14 in the LS of Rubisco. The entire nucleotide sequence for the tobacco (Nicotiana tabacum) Rubisco LSMT (rbcMT-T) gene including the putative promoter region was recently reported, and sequence analysis of the promoter region revealed seven GT-1 motifs. The ability of several truncated rbcMT-T promoter fragments to confer light responsiveness to reporter gene expression in transgenic soybean (Glycine max) hairy roots was examined. Chimeric constructs consisting of the rbcMT-T promoter fused to a bacterial β-glucuronidase (GUS) reporter gene and transferred to soybean via Agrobacterium rhizogenes were evaluated. The rbcMT-T promoter fragments conferred expression of the reporter gene in transgenic hairy roots, callus, and cell suspension cultures based on histochemical and fluorometric GUS analyses. The results suggest a quantitative role for the number of GT-1 motifs in determining differential expression between light and dark conditions. Received: 7 January 1998 / Revision received: 23 March 1998 / Accepted: 13 April 1998     

Phytochemical and physiological changes in Salvia officinalis L. under different irrigation regimes by exogenous applications of putrescine

Water stress is the major factor limiting plant productivity and quality in most regions of the world. In the present study, a two-year field experiment was conducted to determine the influence of putrescine (Put) on phytochemical, physiological, and growth parameters of Salvia officinalis L. under different irrigation regimes. The highest stem dry weight (56.05 and 65.21 g m⁻²) plus leaf dry weight (124.51 g m⁻²) were predicted in irrigation regimes of (20 and 40%) plus 20% available soil water was depleted (ASWD), respectively. Total phenolic content (TPC) was increased significantly under the irrigation regime of 80% with the application of distilled water in spring. TPC showed an increasing trend with increases in Put concentration under all irrigation regimes in both spring and summer. The highest total flavonoids content (TFC) in wavelengths of 415 and 367 nm were predicted in 2.25 mM Put. The highest ascorbate peroxidase (APX) activity (0.13 μmol mg⁻¹ protein) was predicted in the irrigation regime of 20% with the application of distilled water in spring and summer. There was a significantly negative correlation coefficient between APX, TPC, and TFC. Indeed, there was a decreasing trend in APX and an increasing trend in TPC and TFC with increases in Put concentration under the irrigation regime of 20% ASWD. The highest hydroxyl radical scavenging activity (HRSA) values were obtained under irrigation regimes of 49.27% and 20% ASWD in spring and summer, respectively. There was an increasing trend in endogenous Put with increases in the Put concentration. The responses of compatible osmolytes to irrigation regime can be expressed by quadratic model, suggesting maximum proline (0.52 mg g⁻¹), total reducing sugars (TRS) (0.37 mg g⁻¹), xylose (0.68 mg g⁻¹), and mannose (0.37 mg g⁻¹) values would be obtained in irrigation regimes of 68.33%, 48.33%, 53.75%, and 56.25% ASWD, respectively.