Putrescine N-methyltransferase: crystallisation experiments and site directed mutagenesis

BIRGIT DRÄGER
Martin-Luther-Universität Halle-Wittenberg
Institut für Pharmazeutische Biologie und Pharmakologie
Hoher Weg 8
D-06120 Halle (Saale)
birgit.draeger@pharmazie.uni-halle.de
http://ag-bioarznei.pharmazie.uni-halle.de

References
Richter, U., Rothe, G., Fabian A.-K., Rahfeld, B., and Dräger, B. (2005)
Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures. Journal of Experimental Botany, 56, 645-652
The medicinally applied tropane alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna L. and in a small number of other Solanaceae. Calystegines are nortropane alkaloids that derive from a branching point in the tropane alkaloid biosynthetic pathway. In A. belladonna root cultures, calystegine molar concentration is 2-fold higher than that of hyoscyamine and scopolamine. In this study, two tropinone reductases forming a branching point in the tropane alkaloid biosynthesis were overexpressed in A. belladonna. Root culture lines with strong overexpression of the transcripts contained more enzyme activity of the respective reductase and enhanced enzyme products, tropine or pseudotropine. High pseu-dotropine led to an increased accumulation of calystegines in the roots. Strong expression of the tropine-forming reductase was accompanied by 3-fold more hyoscyamine and 5-fold more scopolamine compared with control roots, and calystegine levels were decreased by 30-90% of control. In some of the ransformed root cultures, an increase of total tropane alkaloids was ob-served. Thus,transformation with cDNA of tropinone reductases successfully altered the ratio of tropine-derived alkaloids versus pseudotropine-derived alkaloids.

Brock, A. Bieri, S., Christen, P. and Dräger, B. (2005) Calystegines in wild and cultivated Erythroxylum species. Phytochemistry 66, 1231-1240
Calystegines were identified in the genus Erythroxylum for the first time. Erythroxylum novogranatense var. novogranatense, a species cultivated for cocaine production, contained 0.2% total calystegines in dry leaves. Forty six Erythroxylum herbarium species
consisting mostly of leaf tissue were analysed for calystegines, and 38 were found positive. Calystegines were compared qualitatively and quantitatively between individual Erythroxylum species. Calystegines A3 and B2 were the major calystegines in most species. Total calystegine content reached up to 0.32% dry mass. The simultaneous occurrence of calystegines, cocaine, other alkaloids of a 3a-hydroxy- or 3b-hydroxytropane structure together with nicotine supports the concept of common biosynthetic steps of these alkaloids in Erythroxylum. The present re-sults are the basis for further investigations of the phylogenetic origin of tropane alkaloid biosynthesis in the taxonomically remote families Solanaceae and Erythroxylaceae.2005 Elsevier Ltd. All rights reserved.

Bartholomeusz, T.A.,. Bhogal, R. K, Molinié, R., François-Xavier Felpin, F.-X., Mathé-Allainmat, M., Meier, A.-C., Dräger, B., Lebreton, J., Roscher, A, Robins, R.J., Mesnard, F. (2005) Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved. Phytochemistry 66, 1890-1897
Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nico-tine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [13C,2H3-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-15N]nornicotine re-sulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are me-tabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation

Stenzel, O., Teuber, M., Dräger, B. (2006) Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant. Planta 223, 200-21
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.) contain 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 inves- tigated 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% iden-tity). 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 frag-ments 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 (KM 250 microM) and inhibited by n-butylamine and cadaverine. While spds was transcribed in all plant or- gans, 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 ac-tive.

Biastoff, Stefan, Teuber, Michael, Zhou, Zhaohui Sunny, Dräger, Birgit (2006) Colorimetric activity measurement of a recombinant putrescine N-methyltransferase from Datura stramonium L. Planta Medica 72, 1136-1141
Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the S-adenosyl-L-methionine (SAM or AdoMet)-dependent methylation of putrescine to N-methylputrescine within the biosynthetic pathways of calystegines, nicotine, and tropane alkaloids in medicinal plants and produces S-adenosyl-L-homocysteine (SAH or AdoHcy). Determination of PMT activity was time-consuming and hardly reproducible in the past because it required tedious separation steps after chemical derivatisation or radioactive labelling of N-methylputrescine. A convenient and accurate enzyme-coupled colorimetric assay is based on the conversion of SAH to homocysteine by 5?-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN/SAHN, EC 3.2.2.9) and S-ribosylhomocysteine lyase (LuxS, EC 4.4.1.21). Homocys-teine is quantified by 5,5?-dithiobis-2-nitrobenzoic acid. Putrescine was shown not to interfere with MTAN or LuxS. The colorimetric assay was validated by HPLC analysis. Km values de-termined by the assay, 108 mM for putrescine and 42 mM for SAM, are lower than the previ-ously reported values, due to alleviation of PMT inhibition by SAH.

Kaiser, Kaiser, Richter, Ute, Keiner, Ronald, Brabant, Anja, Hause, Bettina, Dräger, Birgit (2006) Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts.Planta 225, 127-137
Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseu-dotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

Brock, Andrea, Herzfeld, Tobias, Paschke, Reinhard, Koch, Marcus, Dräger, Birgit (2006) Brassicaceae contain nortropane alkaloids. Phytochemistry 67, 2050-2057
The report of cochlearine, the 3-hydroxybenzoate ester of tropine found in Cochlearia officinalis, Brassicaceae, initiated a screening for tropane alkaloids in Cochlearia species and for calystegines in further Brassicaceae. All ten Cochlearia species investigated contained cochlearine, tropine, and pseudotropine. Calystegines, nortropane alkaloids deriving from pseudotropine, were also identified in all Cochlearia species and accumulated up to 0.5% dry mass in leaves. Brassicaceae species of all major lineages of the family were analysed for ca-lystegines. Of the 43 species included in the study, 18 accumulated calystegines of various structures. This is the first screening of Brassicaceae for products of the tropane alkaloid path-way, which is known as characteristic for plants of Solanaceae family. The identification of calystegines in all branches of the Brassicaceae family including Aethionema, a species at the basis of the family, suggests tropane alkaloids as secondary compound typical for Brassicaceae.

Richter, U., Sonnewald, U., Dräger, B. (2007) Calystegines in potatoes with genetically engineered carbohydrate metabolism. Journal of Experimental Botany, 58 1603 - 1615
Calystegines are hydroxylated nortropane alkaloids derived from the tropane alkaloid biosynthetic pathway. They are strong glycosidase inhibitors and occur in vegetables such as potatoes, tomatoes, and cabbage. Calystegine accumulation in root cultures was described to increase with carbohydrate availability. Whether this is indicative for the in planta situation is as yet unknown. Potatoes are model plants for the study of carbohydrate metabolism. Numer-ous transgenic potato lines with altered carbohydrate metabolism are available, but rarely were examined for alterations in secondary metabolism. In this study, calystegine accumulation and expression of biosynthetic enzymes were related to genetic modi.cations in carbohydrate me-tabolism in potato tubers. Tubers contained more soluble sugars due to overexpression of yeast invertase in the apoplast or in the cytosol, or due to antisense suppression of sucrose synthase. It is shown that the major part of calystegines in tubers originated from biosynthesis in plant roots. Yet, tuber calystegine levels responded to genetic alterations of carbohydrate metabolism in tubers. The strongest increase in calystegines was found in tubers with suppressed sucrose synthase activity. Transcripts and enzyme activities involved in calystegine biosynthesis largely concurred with product accumulation. Whole plant organs were examined similarly and displayed higher calystegines and corresponding enzyme activities in roots and stolons of plants with enhanced soluble sugars. Increases in alystegines appear to be linked to sucrose availability.

Teuber M., Meier, A.-C, Azemi, M. E., Namyojan, F., Wodak, A., Brandt, W., Dräger, B. (2007) Putrescine N-methyltransferases - A structure-function analysis. Plant Molecular Biology 63, 787-801
Putrescine N-methyltransferase (PMT) is a key enzyme of plant secondary metabolism at the start of the specific biosynthesis of nicotine, of tropane alkaloids, and of calystegines that are glycosidase inhibitors with nortropane structure. PMT is assumed to have developed from spermidine synthases (SPDS) participating in ubiquitous polyamine metabolism. In this study decisive differences between both enzyme families are elucidated. PMT sequences were known from four Solanaceae genera only, therefore additional eight PMT cDNA se-quences were cloned from five Solanaceae and a Convolvulaceae. The encoded polypeptides displayed between 76% and 97% identity and typical amino acids different from plant sper-midine synthase protein sequences. Heterologous expression of all enzymes proved catalytic activity exclusively as PMT and Kcat values between 0.16 s^-1 and 0.39 s^-1. The active site of PMT was initially inferred from a protein structure of spermidine synthase obtained by protein crystallisation. Those amino acids of the active site that were continuously different between PMTs and SPDS were mutated in one of the PMT sequences with the idea of changing PMT activity into spermidine synthase. Mutagenesis of active site residues unexpect-edly resulted in a complete loss of catalytic activity. A protein model of PMT was based on the crystal structure of SPDS and suggests that overall protein folds are comparable. The respec-tive cosubstrates S-adenosylmethionine and decarboxylated S-adenosylmethionine,
however, appear to bind differentially to the active sites of both enzymes, and the substrate putrescine adopts a different position.

Reviews
Dräger, B. (2006) Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism
Phytochemistry 67, 327-337
Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid me-tab. Products of tropane metab. are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorpo-rated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone re-ductases enabled kinetic characterization, protein crystn., and structure elucidation. Stereo-specificity of redn. is achieved by binding tropinone in the resp. enzyme active center in oppo-site orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tis-sue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the pre-cursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydro-genases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterization. [on SciFinder (R)]

Robins, R. J., Molinié, R., Kwiecien, R. A., Paneth, P., Lebreton, J., Bartholomeusz, T. A., Roscher, A., Dräger, B., Meier, A.-C., Mesnard, F. (2007) Progress in understanding the N-demethylation of alkaloids by exploiting isotopic techniques Phytochem. Reviews 6:51-63
The reaction of N-demethylation plays an important role in the degradation of some alkaloids in a number of organisms. This review presents how our understanding of the N-demethylation of nicotine in plants has been improved through studies in cell cultures of Nicotiana plumbaginifolia and N. glutinosa using a variety of isotopic techniques. The overall aim is to understand how metabolism recycles the alkaloid skeleton, both in terms of the meta-bolic route(s) exploited and the reaction mechanisms of the enzymes involved. The former
has been approached using high-resolution 2-dimensional NMR and GC-MS methods; the
latter by determining kinetic isotope effects and modelling the potential reaction steps. It ap-pears that the mechanism for nicotine demethylation in plants is similar to but has significant
differences from that described for mammals and Pseudomonas bacteria. These differences are discussed.

Bookchapter
Dräger B. (2006) Biotechnology of Solanaceae Alkaloids: A Model or an Industrial Per-spective? In: Kayser, O. and Quax, W., Medicinal Plant Biotechnology, Wiley-VCH Weinheim, Germany

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