iridoids and secologanin

Secologanin, a secoiridoid glucoside, is a pivotal terpenoid intermediate in the biosynthesis of biologically active monoterpenoid indole alkaloids such as reserpine, ajmaline, and vinblastine which are biosynthesized via strictosidine, an alkaloidal glucoside, formed from secologanin and tryptamine. In secologanin biosynthesis, the oxidative cleavage process of loganin to secologanin and the hydroxylation of 7-deoxyloganin to loganin have remained unknown enzymologically and mechanistically. Cornoside is a unique glucoside with 4-hydroxy-2,5-cyclohexadien-1-one (benzoquinol) ring and is widespread in families such as Cornaceae, Oleaceae, and Scrophulariaceae but its biosynthesis, especially the oxidative process, remain to be investigated. Shikonin is a red naphthazarin pigment derived from the roots of Lithospermum erythorhizon and produced biotechnologically by cell cultures. Its biosynthesis including the production regulation mechanism has been investigated in detail. However, the naphthazarin ring formation process, probably starting with the hydroxylation of the side chain of geranylhydroquinone, a key intermediate at the late stage of shikonin biosynthesis, remained unknown. In the present review, cytochrome P450 monooxygenases involved in the biosyntheses of three structurally and biosynthetically interesting compounds, secologanin, cornoside, and shikonin, a described together with the results of previous and recent biosynthetic studies. The biosyntheses of related compounds are also discussed.

Topics: Cyclohexanones; Cytochrome P-450 Enzyme System; Glucosides; Indole Alkaloids; Iridoid Glucosides; Iridoids; Naphthoquinones; Oxidation-Reduction; Plants; Secologanin Tryptamine Alkaloids; Tryptamines; Vinca Alkaloids

In the presence of the enzyme strictosidine synthase, the coupling reaction of secologanin and tryptamine is completely stereoselective and affords strictosidine with 3S configuration, exclusively. The stereoselectivity is transferred and retained in most indole alkaloids of type I in which C-3 is not involved in subsequent reactions. By using results of model reactions, the stereoselectivity was interpreted by the bulkiness of the enzyme temporarily attached to the N-4 atom in the formation of the indolenine intermediate. 3S configuration is kept in the subsequent 1,2-rearrangement into the beta-carboline structure. In the formation of the oxindole derivatives, the 3S configuration is preferred, but not necessarily complete.

Topics: Alkaloids; Carbon-Nitrogen Lyases; Indoles; Iridoid Glucosides; Iridoids; Molecular Conformation; Pyrans; Stereoisomerism; Substrate Specificity; Terpenes

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

Topics: Camptotheca; Cytochrome P-450 Enzyme System; Histidine; Iridoid Glucosides; Iridoids; Mutation; Oxidoreductases Acting on CH-CH Group Donors; Tryptamines

We report the access to an acyclic

Topics: Indole Alkaloids; Iridoid Glucosides; Iridoids; Monoterpenes; Secologanin Tryptamine Alkaloids

To study the chemical composition and their anti-inflammatory activities of honeysuckle (Lonicera japonica Thunb.) roots, seventeen compounds were isolated from the roots of L. japonica Thunb. by various chromatography, including silica gel, Sephadex LH-20 and preparative HPLC. Their structures were identified by MS, IR, and nuclear magnetic resonance spectra, as 1-oxo-(1H)-cyclopenta[b]benzofuran-7-carbaldehyde (1), 4-hydroxycinnamic acid (2), chlorogenic acid (3), loganin aglycone (4), caffeic acid (5), secologanin dimethyl acetal (6), korolkoside (7), coniferin (8), sweroside (9), secoxyloganin (10), 5-O-caffeoylquinic acid (11), chlorogenic acid methyl ester (12), chlorogenic acid ethyl ester (13), 3,5-O-dicaffeoylquinic acid (14), 4,5-O-dicaffeoylquinic acid (15), grandifloroside (16), and 4,5-O-dicaffeoylquinic acid (17). Among those, compound 1 is a new compound, and compound 8 is found in L. japonica for the first time. Compounds 1, 3, 14-17 showed significant anti-inflammatory activities against macrophage in zebrafish.

Topics: Animals; Anti-Inflammatory Agents; Chlorogenic Acid; Chromatography, High Pressure Liquid; Iridoid Glucosides; Iridoids; Lonicera; Macrophages; Plant Roots; Quinic Acid; Zebrafish

During comparative analysis on Palicourea species from Costa Rica, two unusual loganin derived tryptamine-iridoid alkaloids were isolated from an accession of Palicourea crocea. Besides the already known brachycerine (2), palicroceaine (1) features a novel hexacyclic backbone. A second provenance, however, yielded strictosidinic acid (3), belonging to the more common secologanin derived tryptamine-iridoid alkaloids, such as those found in Palicourea padifolia. From this species, strictosidine (4), lyaloside (5) and its derivative (E)-O-(6')-(4″-hydroxy-3″,5″-dimethoxy)-cinnamoyl lyaloside (6) could be isolated. A herbarium specimen-based screening was performed, indicating some degree of regional differentiation in alkaloid content and biosynthetic pathways within the widespread and variable Pal. crocea. It further shows its differentiation from the related strictosidine containing Palicourea croceoides. The occurrence of loganin derived tryptamine-iridoid alkaloids in Pal. crocea, Psychotria brachyceras and Psychotria brachypoda, all putatively unrelated members of the Palicourea s.l. clade, is a noteworthy exception within the genus, otherwise largely characterized by secologanin-derived tryptamine-iridoid alkaloids.

Topics: Costa Rica; Indole Alkaloids; Iridoid Glucosides; Iridoids; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Rubiaceae; Secologanin Tryptamine Alkaloids

Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds.

Topics: Amino Acid Sequence; Biocatalysis; Biosynthetic Pathways; Catharanthus; Cloning, Molecular; Cytochrome P-450 Enzyme System; Gene Silencing; Iridoid Glucosides; Iridoids; Molecular Sequence Data; Oxidation-Reduction; Phylogeny; Plant Proteins; Real-Time Polymerase Chain Reaction; Saccharomyces cerevisiae; Vinca Alkaloids

The manipulation of pathways to make unnatural variants of natural compounds, a process often termed combinatorial biosynthesis, has been robustly successful in prokaryotic systems. The development of approaches to generate new-to-nature compounds from plant-based pathways is, in comparison, much less advanced. Success will depend on the specific chemistry of the pathway, as well as on the suitability of the plant system for transformation and genetic manipulation. As plant pathways are elucidated, and can be heterologously expressed in hosts that are more amenable to genetic manipulation, biosynthetic production of new-to-nature compounds from plant pathways will become more widespread. In this chapter, some of the key strategies that have been developed for metabolic engineering of plant pathways, namely directed biosynthesis, mutasynthesis, and pathway incorporation of engineered enzymes are highlighted. The iridoid-derived monoterpene indole alkaloids from C. roseus, which are the focus of this chapter, provide an excellent system for developing these strategies.

Topics: Biological Products; Biosynthetic Pathways; Carbon-Nitrogen Lyases; Catharanthus; Culture Media; Indole Alkaloids; Iridoid Glucosides; Iridoids; Metabolic Engineering; Monoterpenes; Mutagenesis, Site-Directed; Plant Proteins; Plasmids; Tryptamines; Vinca Alkaloids

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.

Topics: Alkaloids; Biocatalysis; Carbon-Nitrogen Lyases; Catharanthus; Histidine; Iridoid Glucosides; Iridoids; Molecular Chaperones; Oligopeptides; Recombinant Fusion Proteins; Tryptamines; Vinca Alkaloids

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis-enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Topics: Amino Acid Sequence; Catharanthus; DNA, Complementary; Expressed Sequence Tags; Gene Expression Regulation, Plant; Gene Library; Iridoid Glucosides; Iridoids; Kinetics; Methyltransferases; Models, Biological; Molecular Sequence Data; Molecular Structure; Plant Epidermis; Plant Leaves; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Vinblastine; Vinca Alkaloids

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

Topics: Anthranilate Synthase; Catharanthus; Indole Alkaloids; Iridoid Glucosides; Iridoids; Plant Roots; Tryptamines; Tryptophan; Xylulose

On the occasion of having won the Lajos Winkler Medal, at first the author confessed his intellectual relation to the famous scientist. Then he presented his results in the study on the three-dimensional molecular structure and chemotaxonomy of the more than 2500 alkaloids derived from secologanin and tryptamine, and isolated from three plant families, only. The multiple genetic and structural connections among the individual compounds were supported by current and common organic reaction mechanisms. The unified and well articulated picture obtained on detailed search in electronic data bases demonstrated the order of the parts in the whole. The final section of the lecture appreciated the interplay of multiplicity and unity in our culture and education as well.

Topics: Electronic Data Processing; Indole Alkaloids; Iridoid Glucosides; Iridoids; Models, Molecular; Plants; Tryptamines

Plant alkaloids exhibit a diverse array of structures and pharmaceutical activities, though metabolic engineering efforts in these eukaryotic pathways have been limited. Strictosidine synthase (STR) is the first committed step in the biosynthesis of over two thousand terpene indole alkaloids. We describe a rational redesign of the STR binding pocket to selectively accommodate secologanin substrate analogs. The mutant is selective for a substrate that can be chemoselectively derivatized. Evidence that this substrate can be processed by later steps of the terpene indole alkaloid pathway is provided. The work demonstrates that the central enzyme of this alkaloid pathway can be redesigned and that the pathway can turn over the unnatural intermediate that is generated. Modulation of the substrate specificity of enzymes of this complex pathway is therefore likely to enable metabolic engineering efforts of these alkaloids.

Topics: Carbon-Nitrogen Lyases; Indole Alkaloids; Iridoid Glucosides; Iridoids; Mutation; Substrate Specificity

[reaction: see text] Racemic oxodiester 1 undergoes stereoselective cyclocondensation with (S)-tryptophanol, (S)-(3,4-dimethoxyphenyl)alaninol, or the corresponding amino acids, in a process involving a tandem dynamic kinetic resolution/desymmetrization of diastereotopic groups, to give bicyclic lactams, which are cyclized to substituted indolo[2,3-a]- and benzo[a]quinolizidines.

Topics: Amino Acids; Biological Products; Cyclization; Indole Alkaloids; Iridoid Glucosides; Iridoids; Lactams; Molecular Structure; Quinolizines; Stereoisomerism

Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-13C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C]Glc. The in vivo study followed incorporation of [1-13C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-D-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.

Topics: Camptothecin; Carbon Isotopes; Citric Acid Cycle; Computer Simulation; Glucose; Glycolysis; Iridoid Glucosides; Iridoids; Models, Biological; Nuclear Magnetic Resonance, Biomolecular; Pentose Phosphate Pathway; Plant Roots; Rubiaceae; Tryptophan

From the dried roots of Neonauclea sessilifolia, two new chromone-secoiridoid glycosides, sessilifoside and 7"-O-beta-D-glucopyranosylsessilifoside, and three novel indole alkaloid glycosides, neonaucleosides A, B, and C, were isolated along with the main known glycosides, 5-hydroxy-2-methylchromone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, sweroside, loganin, grandifloroside, and quinovic acid 3 beta-O-beta-D-quinovopyranoside-28-O-beta-D-glucopyranoside. The structures of these new glycosides were determined by spectroscopic and chemical means. Neonaucleoside A and its C-3 epimer were prepared from secologanin and tryptamine.

Topics: Glycosides; Indole Alkaloids; Iridoid Glucosides; Iridoids; Nuclear Magnetic Resonance, Biomolecular; Plant Roots; Rubiaceae; Spectrophotometry, Ultraviolet; Stereoisomerism; Tryptamines

The reaction of secologanin (1) (mainly in its tetraacetylated form 1a) with histamine (2) and its benzyl derivative (2b) was investigated. With the benzylated amine (2b), the main product was the normal, tetraacetylated benzyl derivative of histeloside having the R configuration at the new center of chirality, C-1 (5b), with a small amount of an unidentified minor component (probably the 1S epimer 5a). In a slightly acidic medium, the reaction with histamine (2) gave two products in an approximately 6:4 ratio. The main compound proved to be the normal, tetraacetylated derivative of the lactam histelosamide with R configuration at C-1 (7b), and the minor product was the tetraacetylisohisteloside with S configuration at the same C-1 center (3a). When the reaction was carried out under acid-free conditions, in addition to the epimeric pair of the normal tetraacetylated lactam (7a, 7b) and the tetraacetylisohisteloside with 1S configuration (3a), tetraacetylneohistelosamide (8b) was also isolated, in which the cyclization took place at one of the cyclic nitrogens of the imidazole ring. Probably, this latter compound is an intermediate also in the formation of the normal isomers, but under slightly acidic conditions it rapidly isomerized into the normal alkaloid. The tendencies experienced previously in the tryptamine and dopamine series were observed also in the histamine series; that is, at C-1, the R configuration is favored over the S one, and lactamization is faster in the former than in the latter case. The structure of the products and their stereoschemistry were established by NMR spectroscopy.

Topics: Benzene Derivatives; Caprifoliaceae; Chromatography, Thin Layer; Histamine; Iridoid Glucosides; Iridoids; Magnetic Resonance Spectroscopy; Molecular Structure; Plants, Medicinal; Pyrans; Stereoisomerism

The coupling reaction of tetraacetylsecologanin with dopamine and its N-benzyl derivative was investigated. In both series, stereoisomers at C-1, as well as regioisomer normal and neo compounds, were formed. Moreover, the N-unsubstituted products were partially lactamized, and the N-benzyl derivatives epimerized at C-1. In the products, the R configuration of C-1 over the S and the formation of the normal structure over the neo one predominated. The epimerization of both epimers gave an equilibrium of R and S in a ratio of 7:3 and was interpreted by cleavage of the C-1-N-2 bond. The fact that lactamization was much faster in the R than in the S series was explained on the basis of the supposed transition states. The structure, the configuration of C-1, and in several cases the conformations were established by detailed NMR studies and supported by chemical correlations.

Topics: Dopamine; Iridoid Glucosides; Iridoids; Isomerism; Magnetic Resonance Spectroscopy; Pyrans

The coupling reaction of tetraacetylsecologanin with 2,3-dihydro-2-oxotryptamine and its N(b)-benzyl derivative was investigated. With the benzylated amine, the reaction was stopped at the tetracyclic ester level, and with the unsubstituted amine it was immediately followed by lactamization. In both cases, the products were formed with high stereoselectivity at C-3, but as an epimeric pair of 7R and 7S in a ratio of 1:3. The bulky benzyl substituent at N-4 directed the stereoselectivity at C-3 in favor of the S configuration. In the nonbenzylated compounds, the reversible coupling reaction is probably nonstereoselective, but in lactamization the 3R epimer is sterically favored and faster and gives the final lactam in this configuration. The formation of the spiro compounds may serve as a model reaction in the interpretation of the stereoselectivity of the coupling reaction of secologanin with tryptamine in the presence of strictosidine synthase.

Topics: Alkaloids; Benzylamines; Carbon-Nitrogen Lyases; Catalysis; Chromatography, Thin Layer; Cyclization; Iridoid Glucosides; Iridoids; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Structure; Plants, Medicinal; Pyrans; Spiro Compounds; Stereoisomerism; Tryptamines

A new bis-iridoid glucoside, korolkoside (1), was isolated from Lonicera korolkovii, and its structure and stereochemistry were determined by spectroscopic analysis and chemical derivatization. Korolkoside (1) consists of two secologanin moieties that are connected by an acetal linkage.

Topics: Animals; Chromatography, High Pressure Liquid; Glucosides; Iridoid Glucosides; Iridoids; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred Strains; Molecular Conformation; Molecular Structure; Plants, Medicinal; Pyrans; Spectrophotometry, Infrared; Stereoisomerism

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Topics: Biotechnology; Carbon-Nitrogen Lyases; Culture Media; Fruit; Genetic Engineering; Glucosidases; Indole Alkaloids; Iridoid Glucosides; Iridoids; Pyrans; Saccharomyces cerevisiae; Transgenes; Tryptamines

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.

Topics: Carbon Monoxide; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Glucosides; Iridoid Glucosides; Iridoids; Magnoliopsida; Microsomes; NADP; Oxidoreductases Acting on CH-CH Group Donors; Oxygen; Pyrans; Substrate Specificity

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.

Topics: Asteraceae; Cells, Cultured; Cloning, Molecular; Cytochrome P-450 Enzyme System; Escherichia coli; Glucosides; Iridoid Glucosides; Iridoids; Molecular Sequence Data; Oxidoreductases Acting on CH-CH Group Donors; Pyrans; Vinca Alkaloids

Secologanin is the iridoid building block of the majority of the terpenoid indole alkaloids. In the biosynthesis of secologanin, mevalonate was considered to be the exclusive precursor of isopentenyl diphosphate. After [1-(13)C]glucose feeding to a cell culture of Catharanthus roseus, its incorporation into secologanin was studied by 13C NMR spectroscopy. The data showed that the novel triose phosphate/pyruvate and not the mevalonate pathway was the major route for the biosynthesis of secologanin.

Topics: Cells, Cultured; Hemiterpenes; Iridoid Glucosides; Iridoids; Magnetic Resonance Spectroscopy; Molecular Structure; Organophosphorus Compounds; Plants; Pyrans; Pyruvates

Omission of 2,4-D from culture medium during one subculture of Catharanthus roseus cells, strain C20, resulted in an increased alkaloid accumulation, without effect on growth. Alkaloid accumulation, rather than growth, seemed to be more sensitive to 2,4-D. 2,4-D inhibited alkaloid accumulation essentially during growth phase, but its inhibitory effect during this period was partially reversible. As this reversibility was underlined only during the stationary phase, this suggested that this action could be situated upstream in a terpenoid non-specific pathway. 2,4-D feeding showed that inhibition is weaker and weaker as the alkaloid accumulation period proceeds. Auxin action during this period could take place downstream in specific alkaloid pathways. The lower alkaloid accumulation obtained after loganic acid feeding compared to that obtained with secologanin and loganin could indicate that loganic acid methylation should be one of the 2,4-D target(s).

Topics: 2,4-Dichlorophenoxyacetic Acid; Cells, Cultured; Glucosides; Iridoid Glucosides; Iridoids; Plants, Medicinal; Pyrans; Secologanin Tryptamine Alkaloids; Vinca Alkaloids; Yohimbine

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.

Topics: Carbon-Nitrogen Lyases; Cinchona; Indole Alkaloids; Iridoid Glucosides; Iridoids; Plants, Medicinal; Pyrans; Spectrophotometry, Ultraviolet; Transferases; Tryptamines; Vinca Alkaloids