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The biosynthesis of morphine, a stereochemically complex alkaloid, has been shown to occur in plants and animals. A search in the human genome for methyltransferases capable of catalyzing the N-methylation of benzylisoquinoline alkaloids, as biosynthetic precursors of morphine, yielded two enzymes, PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49). Introduction of an N-terminal poly-histidine tag enabled purification of both proteins by immobilized metal affinity chromatography. Recombinant PNMT and NMT were characterized for their catalytic activity towards four benzylisoquinolines: tetrahydropapaveroline (THP), 6-O-methyl-THP, 4′-O-methyl-THP and norreticuline. Human PNMT accepted none of the offered alkaloids and was only active with its established substrate, phenylethanolamine. The second enzyme, human NMT, converted all four benzylisoquinolines, however, with a strict preference for (R)-configured morphine precursors. Determination of kinetic parameters of NMT for the four (R)-configured benzylisoquinoline alkaloids by LC–MS/MS revealed (R)-norreticuline to be the best substrate with an even higher catalytic activity as compared to the previously reported natural substrate tryptamine. In addition, isolation of the morphine precursor salutaridine from urine of mice injected (i.p.) with (R)-THP provides new evidence that the initial steps of morphine biosynthesis in mammals occur stereochemically and sequentially differently than in plants and suggests an involvement of the herein characterized (R)-specific NMT.

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 3' position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine.

It has been firmly established that humans excrete a small but steady amount of the isoquinoline alkaloid morphine in their urine. It is unclear whether it is of dietary or endogenous origin. There is no doubt that a simple isoquinoline alkaloid, tetrahydropapaveroline (THP), is found in human and rodent brain as well as in human urine. This suggests a potential biogenetic relationship between both alkaloids. Unlabeled THP or [ 1,3,4-D 3 ]-THP was injected intraperitoneally into mice and the urine was analyzed. This potential precursor was extensively metabolized (96%). Among the metabolites found was the phenol-coupled product salutaridine, the known morphine precursor in the opium poppy plant. Synthetic [7D]-salutaridinol, the biosynthetic reduction product of salutaridine, injected intraperitoneally into live animals led to the formation of [7D]-thebaine, which was excreted in urine. [ N -CD 3 ]-thebaine was also administered and yielded [ N -CD 3 ]-morphine and the congeners [ N -CD 3 ]-codeine and [ N -CD 3 ]-oripavine in urine. These results show for the first time that live animals have the biosynthetic capability to convert a normal constituent of rodents, THP, to morphine. Morphine and its precursors are normally not found in tissues or organs, presumably due to metabolic breakdown. Hence, only that portion of the isoquinoline alkaloids excreted in urine unmetabolized can be detected. Analysis of urine by high resolution-mass spectrometry proved to be a powerful method for tracking endogenous morphine and its biosynthetic precursors.

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (1) is a key intermediate of the deoxyxylulose phosphate pathway of isoprenoid biosynthesis and a precursor of the plant hormone zeatin. The availability of this intermediate with various labeling patterns is pivotal for its use in biosynthetic studies. The number of positions, however, that can be easily labeled by chemical synthesis is limited, and the synthesis by means of recombinant enzymes is laborious and time consuming. We demonstrated that chromoplasts from Capsicum annuum, whose enzyme activity was impaired by freeze-thawing, accumulate 1. This observation built the basis for the development of a cell-free system allowing the synthesis of this intermediate with labels in various positions. With 2C-methyl-D-erythritol 2,4-cyclodiphosphate (5) as substrate, yields were in the range of 50%.

Root cultures of Tagetes patula L. cv. Carmen were grown with a mixture of unlabeled glucose and [U- 13 C 6 ]glucose or [1- 13 C 1 ]glucose as carbon source. Isoeuparin and (−)-4-hydroxytremetone were isolated by solvent extraction of the cultured tissue, purified by chromatography and analysed by 1 H and 13 C NMR spectroscopy. Amino acids obtained by hydrolysis of protein from the same experiments were used for the reconstruction of the labelling patterns in central metabolic intermediates. These labelling patterns were used for the prediction of isotopolog compositions in the benzofuranone derivatives via different hypothetical pathways. Comparison with the experimentally observed isotopolog distributions showed that the benzenoid ring and the acetoxy group are exclusively or predominantly (>98%) derived from phenylalanine and not from acetyl-CoA via a polyketide-type biosynthesis. The isopropylidene side chain and two carbon atoms of the furan and dihydrofuran moiety, respectively, originate from an isoprenoid building block obtained exclusively or predominantly (>98%) via the deoxyxylulose phosphate pathway. The exomethylene atom of the isopropylidene side chain is biosynthetically equivalent to the ( Z )-methyl group of dimethylallyl diphosphate. The data indicate that isoeuparin and (−)-4-hydroxytremetone are assembled from 4-hydroxyacetophenone and dimethylallyl diphosphate via prenyl-substituted 4-hydroxyacetophenone and dihydrobenzofurans as intermediates.

Experimental and theoretical investigations concerning the second-to-last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4-oxo-DMAPP (12) and 4-hydroxy-DMAPP (11) were synthesized in deuterium- or tritium-labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP (7), 11, and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three-dimensional model of the fragment L271-A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe(4)S(4) cluster, and MEcPP (7) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP (7), hydroxyl abstraction, and formation of HMBPP (8). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe(4)S(4) cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP (8) to IPP (9) and DMAPP (10) in the last step.

High field1H NMR spectroscopy has been employed to obtain, in conjunction with chemometric analysis, information regarding fluctuations in endogenous metabolic profiles forCrotalaria cobalticolaplant cells following exposure to cobalt chloride.Such ‘metabolomic’ type data analysis is often confounded by experimental, environmental or genetic factors that are not correlated to the classifications of interest and serve only to complicate the extraction of meaningful answers from a dataset. This work demonstrates the application of data filtering to remove extraneous data that result from spectrometer variation rather than being correlated with the classes of interest. Samples were analysed fromCrotalaria cobalticolasuspension cell culture following exposure to cobalt chloride using 2 spectrometers. Removal of confounding data due to spectrometer variation resulted in clear separation between control and dosed classes. It was then possible to use the model to determine key changes in biochemical status caused as a result of exposure to cobalt. Branched chain amino acids, succinate and secondary metabolite precursors phenylalanine and tyrosine were all higher in the control samples, whilst choline, glutamate, alanine and lactate were higher in the dosed samples.

For the investigation of the ultimate and penultimate steps of the deoxyxylulose phosphate pathway in plants and microorganisms, and to solve intracellular transport problems, we have developed a facile enzymatic preparation of 2-methyl-D-erythritol 2,4-cyclodiphosphate in highly radioactive form. Use has been made of spinach chloroplast stroma, as well as the stroma of Capsicum annuum and Narcissus pseudonarcissus chromoplasts, which were shown to transform differently labeled 1-deoxy-D-xylulose 5-phosphate quantitatively into that cyclic diphosphate in the presence of cofactors. This method can also be exploited to synthesize milligram quantities of 13C-labeled cyclic diphosphate. It is shown that recombinant Escherichia coli deoxyxylulose phosphate synthase is catalytically active and can be used to synthesize the labeled starting material for further enzymatic work, as well as for feeding of intact plants. The pH and temperature stability of the deoxyxylulose 5-phosphate and the cyclic diphosphate have been determined. Uniformly 13C- or 14C-labeled pyruvate, and uniformly 13C- or 14C-labeled D-glyceraldehyde 3-phosphate were synthesized in almost quantitative yields from uniformly 13C- or 14C-labeled D-glucose en route to deoxyxylulose 5-phosphate.

Plants have been shown to use the mevalonate pathway for the biosynthesis of sterols and triterpenes in the cytoplasm and the recently discovered deoxyxylulose phosphate pathway for the biosynthesis of a variety of hemiterpenes, monoterpenes, diterpenes, as well as for the biosynthesis of carotenoids and the phytol side chain of chlorophyll in plastids. Despite the compartmental separation, at least one terpene precursor can be exchanged between the two pathways. In order to assess quantitatively the crosstalk between the two isoprenoid pathways, [2-13C1]mevalonolactone or [U-13C6]glucose were supplied to cell cultures of Catharanthus roseus grown under illumination or in darkness. Sitosterol, lutein and phytol were isolated and analysed by NMR spectroscopy. The incorporations of exogenous [2-13C1]mevalonolactone were 48% and 7% into the DMAPP and IPP precursors of sitosterol and lutein, respectively. With [U-13C6]glucose as precursor, at least 95% of sitosterol precursors were obtained via the mevalonate pathway, whereas phytol appeared to be biosynthesised via the deoxyxylulose phosphate pathway (approximately 60%) as well via the mevalonate pathway (approximately 40%). The apparent ratios for the contribution of the two pathways depend on the nature of the precursor supplied as well as the nature of the target compound. Thus, crosstalk between the two terpenoid pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms.

An HPLC method was applied for the determination of colchicine and colchicoside in the methanolic extract of the seeds of Colchicum autumnale L. during different stages of seed maturity. The method is carried out isocratically on a reversed-phase column using H2O-CH3CN (95:5) +100 µl H3PO4. The detection limits were 0.10 and 0.05 µg for colchicine and colchicoside, respectively and the recoveries (99.2 ± 1.1%) were high and quantitative. Also, a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitative determination of colchicine, its structurally related compounds (colchicinoids) in seeds of C. autumnale and suspension culture of Colchicum variegatum L. A combined experiment (HPLC- ELISA) was performed for detection of colchicine and some other colchicinoids in C. autumnale seeds extract.

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.

The synthesis of phytochelatins (PCs) in a marine alga, Dunalliela tertiolecta, is strongly induced by Zn. Pretreatment of the cells with Zn enhances the tolerance toward toxic heavy metals such as Cd, Hg, Cu, Pb, and arsenate. Moreover, the pretreatment also increases the tolerance toward oxidative stress caused by hydrogen peroxide or paraquat. In vitro analysis shows that PC is a stronger scavenger of hydrogen peroxide and superoxide radical than glutathione. These results suggest that PCs inducibly synthesized by Zn treatment could play a role not only in detoxification of heavy metals but also in mitigation of oxidative stress.

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)·metal complexes, various other metal·thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451–31459).

2C-methyl-D-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl-D-erythritol 4-phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radiolabelled precursors, we have now demonstrated that [U-13C5]2C-methyl-D-erythritol 4-phosphate affords [U-13C5]2C-methyl-D-erythritol 2,4-cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C-methyl-D-erythritol 4-phosphate as well as 2C-methyl-D-erythritol 2,4-cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2'-13C2]2C-methyl-D-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase.

In a recent report, it was claimed that azuki beans (Vigna angularis) do not synthesize phytochelatins (PCs) upon exposure to cadmium, although glutathione (GSH), the substrate for PC synthesis, is present in this plant. This legume species thus would be the first exception in the plant kingdom that would fail to complex heavy metals by PCs. Here, we report that not GSH, but only homoglutathione can be detected in this plant and that homo-phytochelatins are formed when azuki beans are challenged with heavy metals such as cadmium. We also show that the 5,5′-dithiobis(2-nitrobenzoic acid)-oxidized GSH reductase recycling assay, used for GSH quantification in the recent study of heavy metal tolerance in azuki beans, reacts both with GSH and homoglutathione and therefore cannot be used when biological samples should be analyzed exclusively for GSH.

An analytical strategy based on the sensitivity of electrospray tandem mass spectrometry following a simplified and reproducible sample preparation procedure was evaluated for the determination of Cd-induced phytochelatins (PC) and related peptides in four maize varieties. In addition to the three known families of PC (PC, desGly-PC and iso-PC(Glu)) that were observed, novel PC and desGly-PC homologues lacking the N-terminal gamma-linked Glu were isolated from maize root extracts for the first time. Additionally the complete sequence of iso-PC3(Glu) was determined by tandem mass spectrometry. Peptides obtained in vivo and in vitro as the result of the reaction of glutathione with the enzyme phytochelatin synthase were compared. Minor forms detected from in vitro reactions include compounds with intramolecular or intermolecular disulfide bonds resulting from the oxidation of SH groups, phytochelatin homologues lacking the N-terminal gamma-linked Glu, and new PC-related peptides with a Cys-Cys motif. Since peptides lacking a gammaGlu residue could be generated as artifacts in electrospray mass spectrometry, the application of capillary electrophoresis with online electrospray mass spectrometry allowed the separation and detection of such peptides as endogenous molecules present in planta and as products of in vitro reactions.

The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-13C]glucose or [U-13C6]glucose. 13C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10-terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.

The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-(13)C]glucose or [U-(13)C(6)]glucose. (13)C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. (13)C enrichments and coupling patterns show that the C(10)-terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.

Using nano-electrospray ionization double quadrupole orthogonal-acceleration time-of-flight mass spectrometry, we were able to detect sub-nanogram levels of endogenous morphine and its metabolite morphine-6-glucuronide in the rat pheochromocytoma cell line. Furthermore, fragmentation of endogenous morphine-6-glucuronide (462.14 Da) by collision-induced dissociation yields morphine (286.14 Da) obtained from the cell samples and with the authentic material, further demonstrating the presence of endogenous morphine. The incubation medium was devoid of this material. This methodology represents a sensitive and accurate method to detect trace amounts of endogenous opiates at subnanogram levels in biological samples.

1-deoxy-D-xylulose 5-phosphate serves as a precursor for the biosynthesis of the vitamins thiamine and pyridoxal and for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate via the nonmevalonate pathway of terpenoid biosynthesis. Earlier studies had shown that Escherichia coli incorporates unphosphorylated 1-deoxy-D-xylulose into the terpenoid side chain of ubiquinones with high efficacy. We show that D-xylulokinase of E. coli (EC 2.7.1.17) catalyzes the phosphorylation of 1-deoxy-D-xylulose at the hydroxy group of C-5 at a rate of 1.6 micromol.mg min-1. This reaction constitutes a potential salvage pathway for the generation of 1-deoxy-D-xylulose 5-phosphate from exogenous or endogenous 1-deoxy-D-xylulose as starting material for the biosynthesis of terpenoids, thiamine and pyridoxal.

The putative catalytic domain (residues 81–401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2- C -methyl- d -erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2- C -methyl- d -erythritol at a rate of 33 μmol⋅mg −1 ⋅min −1 . The structure of the reaction product, 4-diphosphocytidyl-2- C -methyl- d -erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg 2+ , are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78–202 bp. A fragment comprising amino acid residues 76–302 was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl- d -erythritol from 2C-methyl- d -erythritol 4-phosphate with a specific activity of 67 μmol⋅min −1 mg −1 . The Michaelis constants for 4-diphosphocytidyl-2C-methyl- d -erythritol and CTP were 500 μM and 114 μM, respectively.

In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP , which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate into 2C-methyl- d -erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using 13 C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn 2+ or Mg 2+ but no other cofactors. Radioactivity from [2- 14 C]2C-methyl- d -erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl- d -erythritol into 2C-methyl- d -erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.

1-Deoxy-D-xylulose-5-phosphate is converted into 2-C-methyl-D-erythritol-4-phosphate by the catalytic action of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr protein) using NADPH as cofactor. The stereochemical features of this reaction were investigated in in vitro experiments with the recombinant Dxr protein of Escherichia coli using (4R)- or (4S)-[4-(2)H(1)]NADPH as coenzyme. The enzymatically formed 2-C-methyl-D-erythritol-4-phosphate was isolated and converted into 1,2:3,4-di-O-isopropylidene-2-C-methyl-D-erythritol; NMR spectroscopic investigation of this derivative indicated that only (4S)-[4-(2)H(1)]NADPH affords 2-C-methyl-D-erythritol-4-phosphate labelled exclusively in the H(Re) position of C-1. Stereospecific transfer of H(Si) from C-4 of the cofactor identifies the Dxr protein of E. coli as a class B dehydrogenase.

Two specific cytochrome P-450 enzymes were detected in cell suspension cultures of Marchantia polymorpha; the first catalyzes the coupling of two molecules of lunularic acid to form marchantin C and CO2 and the second hydroxylates marchantin C to marchantin A. Cell free experiments using 3 H= 14 C doubly-labeled substrates demonstrated that lunularic acid, and neither lunularine nor prelunularic acid, is the sole substrate for the coupling reaction. Both enzymes are dependent on the presence of oxygen and NADPH. Both reactions were inhibited in the presence of CO in the dark; this inhibition was partially reversed by white light. In addition, both reactions were inhibited by typical inhibitors of cytochrome P-450 enzymes such as cytochrome c and ancymidol. # 1999 Elsevier Science Ltd. All rights reserved.

Cell cultures of the plant Rubia tinctorum were grown with supplements of [1-13C]- or [U-13C6]glucose. Amino acids were obtained by hydrolysis of biomass, and their 13C labeling patterns were used ...

The monoterpene loganin serves as a precursor in the biosynthetic pathways of numerous indole alkaloids. In contrast to earlier studies, we present evidence that the biosynthesis of loganin in Rauwolfia serpentina cells proceeds mainly via the deoxyxylulose pathway and not by the mevalonate pathway. This conclusion is based on experiments using a R. serpentina cell culture supplied with 13 C -labeled samples of glucose, ribose/ribulose, pyruvate or glycerol. Loganin was isolated from biomass, and the hydrolysis of cellular protein afforded amino acids. The isolated metabolites were analyzed by NMR spectroscopy. The 13 C -labeling patterns of isolated amino acids were then used to reconstruct the labeling patterns of phosphoenol pyruvate, pyruvate and acetyl CoA. These labeling patterns were subsequently used to predict labeling patterns for dimethylallyl pyrophosphate and isopentenyl pyrophosphate via the mevalonate and deoxyxylulose pathway, respectively. The observed labeling patterns of the terpenoid moieties in loganin were in excellent agreement with the deoxyxylulose prediction. The minor incorporation of mevalonate into loganin observed in earlier studies can be attributed to metabolite exchange between the two terpenoid pathways. The possibility of crosstalk between the two pathways in plants and plant cell cultures stresses the need for a quantitative analysis of general carbon metabolism in order to determine the partitioning between the mevalonate and deoxyxylulose pathway. The present study shows that a wide variety of general metabolic precursors can fulfill this task in conjunction with the retrobiosynthetic concept.

The presence of the deoxyxylulose pathway in chromo- and chloroplasts of various higher plants was demonstrated by the conversion of labelled 1-deoxy-D-xylulose 5-phosphate into β-carotene and geranylgeraniol, in the presence of ATP. The antibiotic fosmidomycin inhibited the formation of terpenoids from 1-deoxy-D-xylulose 5-phosphate, whereas it did not affect the conversion of 2- C -methyl-D-erythritol 4-phosphate or isopentenyl diphosphate into terpenoids.

An O -acetyltransferase that catalyzes the regiospecific acetylation of a range of taxanes possessing an unsubstituted 10-hydroxyl group was detected and purified to apparent electrophoretic homogeneity from a cytosolic fraction of Taxus chinensis cell cultures. The purification involved negative calcium phosphate adsorption, sephadex desalting, DEAE, AcA44 chromatography, HighQ, CHT II, HiTrap Blue, Phenylsepharose and Mimetic Green purification steps. The purified acetyltransferase was found to be a monomeric protein of 71±1.5 kDa that is highly regio- and stereospecific towards the 10 β -hydroxyl group of the taxane molecule and is also active towards 10-desacetylbaccatine III. The acetyltransferase reaction had a pH optimum of 9.0 with halfmaximal activities at pH 6.8 and 10.8, respectively. The temperature optimum was at 35°C and the isoelectric point at 5.6. The apparent K m values for 10-desacetyltaxuyunnanine C and acetyl CoA were 23 and 61 mM, respectively. The turnover rate for the enzyme using both substrates was 0.2 mol mol −1 of enzyme. The kinetic optimum was determined to be K cat / K m =8.7 s −1 L M −1 .

Using sterile thallus tissue of Marchantia polymorpha , the biosynthesis of the cyclic bis(bibenzyl) marchantin A was investigated. The synthesis of the radioactively and 13 C-labelled precursors for the application experiments is described. Feeding experiments showed that rings A and C of the marchantin molecule are derived from the benzene ring of l -phenylalanine via trans -cinnamic acid and p -coumaric acid. Further application of 13 C-labelled precursor with subsequent 13 C NMR spectroscopy proved that dihydro- p -coumaric acid is an intermediate in marchantin biosynthesis. A phenylpropane/polymalonate pathway using dihydro- p -coumaric acid and acetate/malonate is proposed for the biosynthesis of the bibenzyl monomers which were confirmed to be the building blocks of the marchantin molecule. The bibenzyls in turn are coupled in a unique way to form the bis(bibenzyl) structure.

Application of radioactively as well as 13C-labelled 4′-O-methylnorbelladine to organs of field grown Leucojum aestivum plants resulted in an optimal incorporation into galanthamine (27%), and N-demethylgalanthamine (31%), respectively. In contrast to expectations based on results from the literature, the N-methylated 4′-O-methylnorbelladine was metabolized into a minor extent in L.aestivum and was incorporated into galanthamine as well as into N-demethylgalanthamine to only about 1\3 of the rate of 4′-O-methylnorbelladine. Furthermore, it was shown that N-demethylgalanthamine is N-methylated to galanthamine in the final step of biosynthesis. A revised scheme for the biosynthesis of galanthamine is presented involving the phenol oxidative coupling of 4′-O-methylnorbelladine to a postulated dienone which undergoes spontaneous closure of the ether bridge to yield N-demethylnarwedine which upon stereoselective reduction and N-methylation yields the cholineesterase inhibitor galanthamine.

A cell culture of Taxus chinensis was found to produce taxoids in 4% yield (dry weight) after stimulation with methyl jasmonate. From the culture medium and cell mass, 16 taxoids were isolated. The structures were determined by two-dimensional gradient-enhanced DQF-COSY, HMQC, HMBC, and NOESY NMR experiments. The major taxoids of the culture were esterified at C-14 of the taxoid ring system. Five new taxoids were identified as minor metabolites and were assigned as 2 α -benzoxy-4 α ,10 β -diacetoxy-1 β ,7 β ,9 α -trihydroxytax-11-ene, 2 α -benzoxy-4 α -acetoxy-1 β ,7 β ,10 β -trihydroxy-9-dehydrotax-11-ene, 2 α -benzoxy-4 α ,7 β , 9 α ,10 β -tetraacetoxy-1 β -hydroxytax-11-ene, 2 α ,4 α ,7 β ,9 α ,10 β -pentaacetoxytax-11-ene and 2 α ,5 α ,7 β ,9 α ,10 β -penta- acetoxy-4 β ,20-epoxytax-11-ene.

Leaves of Liriodendron tulipifera convert 1-deoxy-D-xylulose to 2-C-methyl-D-erythritol via a skeletal rearrangement reminiscent of the formation of terpene precursors from 1-deoxy-D-xylulose. The data suggest that 2-C-methyl-D-erythritol 4-phosphate is either an intermediate or a side product of the deoxyxylulose 4-phosphate pathway of terpenoid biosynthesis.

Using an optimized precursor feeding system, the biosynthesis of the alkaloid lunarine in Lunaria annua seeds was investigated. The synthesis of radioactively labelled precursors for the application experiments is described. Lunarine was shown to be synthesized by stereo-selective phenol-oxidative coupling of N 1 , N 10 -bis( p -coumaroyl)spermidine. p -Coumaric acid for the biosynthesis of this dimer is formed from l -phenylalanine via trans -cinnamic acid. The polyamine moiety of lunarine, spermidine, and its immediate precursor putrescine are preferentially synthesized from arginine. Enzyme studies with microsomes of Lunaria annua seeds indicated that a cytochrome P-450 enzyme might be responsible for the phenol-oxidative coupling of N 1 , N 10 -bis ( p -coumaroyl)spermidine yielding the hexahydrodibenzofuran ring of lunarine.

In order to distinguish between ortho-para and para-para phenol coupling in colchicine biosynthesis, [3′-O 14 CH 3 ]autumnaline was converted to [ 14 C]colchicine in seeds of Colchicum autumnale and the product was then selectively demethylated by a bacterium that exclusively demethylates C-3 of colchicine. The retention of the radioactive label in 3-demethylcolchicine indicates that biosynthesis proceeds through para-para coupling.

A Papaver somnifverum cell culture transforms thebaine into tetrahydrothebaine and thebainone while Mahonia nervosa transforms thebaine into oripavine

The incorporation of [1- 13 C]- and [2,3,4,5- 13 C 4 ]1-deoxy- d -xylulose into β-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13 C Enrichment and 13 C 13 C coupling patterns showed conclusively that 1-deoxy- d -xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, β-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Incorporation of [1-13C]- and [U13C6]glucose shows that monoterpenoid essential oils (geraniol, menthone, pulegone, thymol) are biosynthesised in plants by a pathway which is different from the established mevalonic acid route.

Two novel enzyme activities involved in the condensation of dopamine and secologanin were discovered in the cell-free extracts prepared from the leaves of Alangium lamarckii . The extracts condensed dopamine and secologanin rapidly at pH 7.5 to form both (1 R )-deacetylipecoside ( 1 ) and (1 S )-deacetylisoipecoside ( 2 ) which were converted spontaneously to demethylalangiside ( 3 ) and demethylisoalangiside ( 4 ), respectively. Indentification of the enzymatic reaction products was performed by using TLC, HPLC-photodiode array detector and LC-MS. A simple enzyme assay by a spectrophotometric method was developed for enzyme activity detection during enzyme purification. The discovery of these two enzyme activities in A. lamarckii suggested that the naturally occurring ( S )- and ( R )-forms of various tetrahydroisoquinoline-monoterpene alkaloids and nitrogenous glucosides found in this plant are determined by the first enzymatic step of dopamine and secologanin condensation which yields either the (1 R ) or the (1 S )-condensation product.

The alkaloid lunarine occurring in Lunaria annua seeds was shown to be synthesized by stereoselective phenoloxidative coupling from synthetic bis(p-coumaroyl)spermidine. © 1997 Elsevier Science Ltd.

Three distinct Cd-phytochelatin complexes were purified to apparent homogeneity by gel filtration on Sephadex G50 of crude Cd-phytochelatin preparations isolated from Cd-treated suspension cultures of Rauvolfia serpentina . These purified complexes were analysed and found to be significantly different in PC chainlength, S:Cd ratio, and sulphide content. Kinetic measurements of PC n and sulphide content in Cd-treated suspension cultures of R. serpentina and Silene cucubalus showed that there are two possible ways of decreasing the amount of sulphur (and nitrogen) needed for Cd 2+ sequestration: either by increasing the PC chainlength or by incorporating sulphide into the complexes. The latter process appears to be the more effective.

A wide range of compounds derived from the basic structure of jasmonic acid, when tested for biological activity in different bioassays (Eschscholzia californica elicitation, Bryonia dioica tendril coiling, tomato transpiration and senescence and barley senescence assays) display, in each of the assays, an activity profile that is distinctly characteristic and different between assays. While differences in uptake, metabolism and/or sequestration of the compounds may account for some of the effects observed, the data allow the conclusion that structural requirements are also different for different physiological responses regulated by jasmonates. While jasmonic acid itself is active in all assays employed, some of the compounds tested in our study display a much narrower range of biological effects. Thus, tailoring of jasmonate analogues for specific applications and lacking undesirable side effects should be possible.

Enzyme-Linked Immunosorbent Assay (ELISA) was established to qualitatively and quantitatively determine galanthamine in the alcoholic extract of Pancratium foetidum Pomel bulbs and in the callus culture of P. arabicum Sick. The amount of galanthamine was 168 ng/g dry weight of bulbs and 314 ng/g dry weight of the callus. The measuring range for galanthamine extended from 0.1 – 100 ng per assay. The least amount to be detected was 0.01 ng and the recovery was 98 ±1.8 % .

A highly substrate-specific microsomal-bound cytochrome P-450 NADPH and O2-dependent enzyme system has been discovered in immature seeds of Colchicum autumnale L. that is responsible for the intramolecular phenol-oxidative coupling of autumnaline to isoandrocymbine during colchicine biosynthesis.

A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524].

A set of heavy-metal-complexing peptides was isolated from plants and plant suspension cultures. The structure of these peptides was established as (gamma-glutamic acid-cysteine)n-glycine (n = 2-11) [(gamma-Glu-Cys)n-Gly]. These peptides appear upon induction of plants with metals of the transition and main groups (Ib-Va, Z = 29-83) of the periodic table of elements. These peptides, called phytochelatins (PC), are induced in all autotrophic plants so far analyzed, as well as in select fungi. Some species of the order Fabales and the family Poaceae synthesize aberrant PC that contain, at their C-terminal end, either beta-alanine, serine or glutamic acid. For this group of peptides the name iso-PC is proposed. The biosynthesis of PC proceeds by metal activation of a constitutive enzyme that uses glutathione (GSH) as a substrate; this enzyme is a gamma-glutamylcysteine dipeptidyl transpeptidase which was given the trivial name PC synthase. It catalyzes the following reaction: gamma-Glu-Cys-Gly + (gamma-Glu-Cys)n-Gly-->(gamma-Glu-Cys)n+1-Gly + Gly. The plant vacuole is the transient storage compartment for these peptides. They probably dissociate, and the metal-free peptide is subsequently degraded. Sequestration of heavy metals by PC confers protection for heavy-metal-sensitive enzymes. The isolation of a Cd(2+)-sensitive cadl mutant of Arabidopsis thaliana, that is deficient in PC synthase, demonstrates conclusively the importance of PC for heavy metal tolerance. In spite of the fact that nucleic acid sequences and proteins are found in higher plants that have distant homology to animal metallothioneins, there is absolutely no experimental evidence that these "plant metallothioneins' are involved in the detoxification of heavy metals. PC synthase will be an interesting target for biotechnological modification of heavy metal tolerance in higher plants.