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M H Zenk

Publications and source records attributed to M H Zenk.

At least 37 records · Page 2Linked to original sources
PubMed1999

Purification and characterization of acetyl coenzyme A: 10-hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxus chinensis.

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 beta-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 degrees C and the isoelectric point at 5.6. The apparent K(m) values for 10-desacetyltaxuyunnanine C and acetyl CoA were 23 and 61 microM, 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 Kcat/K(m) = 8.7 s-1 L M-1.

Acetyltransferases↗
PubMed1999

Biosynthesis of Erythrina alkaloids in Erythrina crista-galli.

A precursor application system was developed to allow the study of Erythrina alkaloid formation in Erythrina crista-galli. Fruit wall tissue of this species was recognized as the major site of alkaloid biosynthesis. The application of radioactively and 13C-labelled potential precursors showed that the hitherto assumed precursor (S)-norprotosinomenine was not incorporated into the Erythrina alkaloids. In contrast, (S)-coclaurine as well as (S)-norreticuline were metabolized to erythraline and erythrinine, respectively, suggesting that a coclaurine-norreticuline pathway is operative in Erythrina alkaloid formation. Feeding of [1-13C]-labelled (S)-norreticuline with subsequent NMR spectroscopy demonstrated that the resulting erythraline was exclusively labelled at position C-10. Therefore, the participation of a symmetrical intermediate of the diphenoquinone type in Erythrina alkaloid biosynthesis can be excluded.

Alkaloids↗
PubMed1999

Biosynthesis of bitter acids in hops. A (13)C-NMR and (2)H-NMR study on the building blocks of humulone.

The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope-incorporation experiments using (13)C-labelled glucose or (2)H(2)O. (13)C enrichments, (2)H enrichments and (13)C(13)C coupling patterns identify isovaleryl-CoA, malonyl-CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.

Acyl Coenzyme A↗
PubMed1998

(-)-2C-methyl-D-erythrono-1,4-lactone is formed after application of the terpenoid precursor 1-deoxy-D-xylulose.

Application of [1,2-14C]1-deoxy-D-xylulose, the committed precursor of terpenoids, thiamine and pyridoxol, to a variety of plant species resulted in the labelling of an unknown metabolite. The isolation and purification of this metabolite from Ipomoea purpurea plants fed with 1-deoxy-D-xylulose (DX), followed by NMR analysis, resulted in the identification of its structure as (-)-2C-methyl-D-erythrono-1,4-lactone (MDEL). MDEL has been previously isolated as a stress metabolite of certain plants. A hypothetical biosynthetic scheme is given.

Lactones↗
PubMed1998

Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol.

A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.

Alkyl and Aryl Transferases↗
PubMed1998

Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata.

The nickel content in different parts of the hyperaccumulating tree Sebertia acuminata was analysed by atomic absorption spectroscopy. Nickel was found to be mainly located in laticifers. The total nickel content of a single mature tree was estimated to be 37 kg. By gel filtration and NMR spectroscopy, citric acid was unequivocally identified as counter ion for about 40% of this metal present. Nitrate was assumed to be a further partner for a complete ionic balance. Phytochelatins were not found to be involved in nickel detoxification in Sebertia. The localization of nickel complexes inside the laticifers was demonstrated by light microscopy as well as by scanning electron microscopy in combination with an EDX system for the analysis of elements. A repellent effect of the plant sap was observed on the fruit fly Drosophila melanogaster indicating that in hyperaccumulating plants nickel functions as an agent to prevent predation.

Animals↗
PubMed1997

Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement.

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-D-xylulose into beta-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. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-D-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, beta-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.

Cells, Cultured↗
PubMed1996

Heavy metal detoxification in higher plants--a review.

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.

Aminoacyltransferases↗
PubMed1996

Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin.

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].

Cells, Cultured↗
PubMed1995

Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from papaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebaine in morphine biosynthesis.

Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase, a highly substrate-specific enzyme, has been purified nearly 3,000-fold to homogeneity from Papaver somniferum plant cell suspension cultures. Purification was achieved by fractionated ammonium sulfate precipitation, dye-ligand affinity chromatography on matrex red A, gel filtration, ion exchange chromatography on Mono Q and a second dye-ligand affinity chromatography on fractogel TSK AF Blue. The purified enzyme was a single polypeptide with an M(r) = 50,000 displaying an isoelectric point of 4.8, a pH optimum between pH 6 and 9 and a temperature optimum at 47 degrees C. The Km values for the substrate salutaridinol and the co-substrate acetyl co-enzyme A were 7 and 46 microM, respectively. Salutaridinol-7-O-acetyltransferase catalyzes the stoichiometric transfer of the acetyl group from acetyl coenzyme A to the 7-OH group of salutaridinol yielding salutaridinol-7-O-acetate, which is a new intermediate in morphine biosynthesis. Salutaridinol-7-O-acetate undergoes a subsequent spontaneous allylic elimination at pH 8-9, leading to the formation of thebaine (1), the first morphinan alkaloid with the complete pentacyclic ring system, or at pH 7 leading to dibenz[d,f]azonine alkaloids that contain a nine-membered ring. Acetylation and subsequent allylic elimination is a new enzymic mechanism in alkaloid biosynthesis, which in the poppy plant can transform one precursor into alkaloids possessing markedly different ring systems, depending on the reaction pH.

Acetyltransferases↗
PubMed1995

Evidence against specific binding of salicylic acid to plant catalase.

It was demonstrated that salicylic acid (SA) not only binds to catalase from differentiated higher plants and plant cell suspension cultures but also to those of fungi and animals. SA bound specifically to iron-containing enzymes, such as catalase, aconitase, lipoxidase and peroxidase, while not to iron-free plant enzymes. On the grounds of these experiments, the claim is further challenged that SA is a signalling compound and second messenger in plants that activates plant defense-related genes through elevated H2O2 levels by specifically inhibiting catalase activity. SA may just function as a phytoalexin.

Aconitate Hydratase↗
PubMed1995

Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell cultures and differentiated plants.

Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (-)codeinone to (-)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver somniferum L. cell cultures. The purification involved ammonium sulfate precipitation (40-80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 55S), affinity chromatography (fractogel TSK AF Blue), ion-exchange chromatography (DEAE-Sephacel) and native PAGE. The purified codeinone reductase was found to be a monomeric protein of 35 +/- 1 kDa that is highly substrate-specific, reducing only the C6 oxo group of codeinone and morphinone as well as a few analogues. The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1. The temperature optimum is at 40 degrees C and the isoelectric point (p1) at 4.4. The apparent Km values (forward reaction) for codeinone and NADPH are 23 microM and 168 microM, respectively. Using capsule tissue of differentiated P. somniferum plants as an enzyme source, two codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity. These isoenzymes could not be separated for characterization and showed slightly different kinetic features (Km values: codeinone 9 microM; NADPH 81 microM) compared with the cell culture enzyme.

Alcohol Oxidoreductases↗
PubMed1995

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.

Animals↗
PubMed1994

Was opium known in 18th dynasty ancient Egypt? An examination of materials from the tomb of the chief royal architect Kha.

Examination by microscopy, thin-layer chromatography, gas-liquid chromatography alone and combined with mass spectrometry, and radioimmunoassay methods of materials from the tomb of the ancient Egyptian chief royal architect Kha, who is believed to have died about 1405 BC, has shown that there is no morphine--and hence no opium--present. This finding casts doubt on the results of an earlier analysis. Tropane alkaloids are likewise absent. The significance of the present findings for the history of the opium poppy, Papaver somniferum L., in the eastern Mediterranean region is discussed. Evidence (chemical, botanical, artefactual, and linguistic) for the supposed presence of the opium poppy and opium in Egypt in the Late Bronze Age is briefly reviewed. These considerations and the negative outcome of the present analyses mean that the earlier reported finding can no longer be accepted as evidence.

Egypt, Ancient↗
PubMed1994

Immunoassays for the quantitative determination of colchicine.

A radioimmunoassay as well as an enzyme immunoassay for the quantitation of fmol amounts of the alkaloid colchicine have been developed. The antiserum used for both assays was raised against a conjugate of colchicoside-bovine serum albumin. The crude serum was satisfactory for the performance of the radioimmunoassay. For the enzyme immunoassay, the antibodies had to be isolated and purified by Rivanol treatment with subsequent (NH4)2SO4 precipitation. The measuring range extends from 0.1 to 100 ng colchicine for the radioimmunoassay and from 0.05 to 350 ng for the enzyme immunoassay with detection limits of 125 fmol and 25 fmol, respectively. Both immunoassays cross reacted with colchicoside and 3-demethyl-colchicine up to 80%. The colchicine content in the newly established suspension culture of Colchicum variegatum as well as the influence of various culture media on the colchicine production of this cell culture were investigated with the radioimmunoassay. The enzyme immunoassay was well suited for the quantitation of colchicine in HPLC fractions of Gloriosa and Colchicum seed extracts allowing the rapid, sensitive, and precise determination of the substance under investigation. The preliminary experiments indicate that both colchicine immunoassays can be a useful tool for the analysis of colchicine in tissue and cell culture studies, for analysis of plant extracts as well as for biosynthetic investigations.

Animals↗
PubMed1993

Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid.

Fungal cell walls and fragments thereof (elicitors) induce the formation of low and high molecular weight defense compounds in plant cell suspension cultures. This induced synthesis requires a signal molecule transmitting the message between the elicitor plant cell wall receptor and gene activation. We demonstrate in this study that cis-jasmonic acid is rapidly synthesized in plant cell cultures of diverse taxonomic origin (gymnosperms and mono- and dicotyledonous plants) after challenge with a fungal elicitor preparation. The rapid decline of cis-jasmonic acid in some of these tissues is attributed to rapid metabolism of this pentacyclic acid. The induction of alkaloids by several different molecules provoking the elicitation process is strictly correlated with the synthesis of jasmonates. Elicitation leads to a rapid release of alpha-linolenic acid from the lipid pool of the plant cell. alpha-Linolenic acid and 12-oxophytodienoic acid, the formation of which is also induced, are known to be distant precursors of jasmonic acid. We assume cis-jasmonic acid and its precursors to be the signaling molecules in the elicitation process.

Journal Article↗