Ergot Alkaloids and their Hallucinogenic Potential in Morning Glories.

Naturally occurring and semisynthetic ergot alkaloids play a role in health care or as recreational drugs in Western and indigenous Mexican societies. Evidence is summarized that ergot alkaloids present in Central American Convolvulaceae like Turbina corymbosa, Ipomoea violacea, and Ipomoea asarifolia are colonized by different species of a newly described clavicipitaceous fungal genus named Periglandula. The fungi are associated with peltate glandular trichomes on the adaxial leaf surface of its host plants. The Periglandula fungi are not yet culturable in vitro but were demonstrated to have the capacity to synthesize ergot alkaloids. The alkaloids do not remain in the fungal mycelium but are translocated via the glandular trichomes into their plant host. Both fungi and host benefit from a symbiotic lifestyle. In evolutionary terms the alkaloid biosynthetic gene cluster in the Periglandula/Ipomoea symbiosis is likely to have a conserved (basic) structure while biosynthetic ergot gene clusters within the genera Claviceps and Epichloe were under ecological selection for alkaloid diversification.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae.

We describe two newly discovered fungi living on the adaxial leaf surface of plants belonging to the Convolvulaceae, Ipomoea asarifolia and Turbina corymbosa. The fungi apparently are epibionts because hyphae were never observed to penetrate epidermal cells or stomata of their respective host plants, and most remarkably are intimately associated with secretory glands on the leaf surface. Hyphae and structures resembling chlamydospores and synnemata (but lacking conidia), formed by both fungal species are phenotypically nearly indistinguishable after in vitro growth or when examined in vivo on the leaf surface. Phylogenetic trees based on aligned DNA sequences from nuclear genes for ß-tubulin (tubB) and RNA Polymerase II subunit 1 (rpbA), and the mitochondrial gene for ATP synthase F0 subunit A (atp6), grouped the fungal species in a clade within the Clavicipitaceae. Clavicipitaceous fungi isolated from the two different plant species could be distinguished by their atp6 and rpbA sequences, and nuclear genes for γ-actin (actG), translation elongation factor 1-α (tefA), and 4-(γ,γ-dimethylallyl)tryptophan synthase (dmaW), the determinant step in ergoline (i.e. ergot) alkaloid biosynthesis. Based on these findings we propose a new fungal genus, Periglandula, gen. nov., and describe two new species, Periglandula ipomoeae sp. nov., from host plant I. asarifolia, and Periglandula turbinae sp. nov., from T. corymbosa.

Ginkgo biloba and ginkgotoxin.

Products prepared from Ginkgo biloba are top-selling phytopharmaceuticals especially in Europe and major botanical dietary supplements in the United States. In European medicine, G. biloba medications are used to improve memory, to treat neuronal disorders such as tinnitus or intermittent claudication, and to improve brain metabolism and peripheral blood flow. The whole array of indications is reflected by a number of defined natural product constituents in G. biloba. The most well-known ones are flavonoids and terpene lactones, but they also include allergenic and toxic compounds such as ginkgotoxin (1). Consequently, there are reports attributing beneficial as well as adverse effects to G. biloba products. The present paper summarizes recent experiences with G. biloba and its derived products and explains why their restricted use is recommended.

Influence of antivitamins ginkgotoxin 5'-phosphate and deoxypyridoxine 5'-phosphate on human pyridoxine 5'-phosphate oxidase.

The pharmacological effects of leaf extracts (EGb 761) from Ginkgo biloba L. are attributed to ginkgolides, bilobalide and biflavonoids. However, besides these beneficial attributes, ginkgotoxin, a B(6) antivitamin which may cause epileptic convulsions, other severe neuronal disorders and even death, is also found in Ginkgo leaves and leaf-derived remedies. Because of its structural similarity to the B(6) vitamers, an interaction of ginkgotoxin with enzymes involved in the vitamin B(6)-dependent metabolism of the human brain is possible. This led us to investigate how the neurotoxic ginkgotoxin acts in the brain. To this end the gene coding for the human pyridoxine 5'-phosphate oxidase was heterologously overexpressed in E. COLI and the homogeneous enzyme was characterized. The investigation showed that the enzyme is inhibited in vitro by the synthetic vitamin B(6) derivative 4'-deoxypyridoxine 5'-phosphate but not by ginkgotoxin or its 5'-phosphate.

The Pdx1 family is structurally and functionally conserved between Arabidopsis thaliana and Ginkgo biloba.

Vitamin B6 is one of the most important compounds in living organisms, and its biosynthesis has only recently been understood. Because it is required for more than 100 biochemical reactions, lack of the vitamin is fatal. This is of special importance to mammals and humans, which cannot biosynthesize the vitamin and thus depend on its external uptake. Here we describe the cloning of a vitamin B6 biosynthetic gene GbPDX1 from Ginkgo biloba. The gene is expressed in seeds, leaf and trunk tissue. Using yeast 2-hybrid and pull-down assays, we show that the protein can interact with itself and with members of Arabidopsis thaliana AtPDX1 and AtPDX2 families. Furthermore, we prove the function of GbPDX1 in vitamin B6 biosynthesis by complementation of an Arabidopsis AtPDX1.3 mutant rsr4-1, at the phenotypical level and increasing vitamin B6 levels caused by ectopic GbPDX1 expression in the mutant background. Overall, this study provides a first description of Ginkgo vitamin B6 metabolism, and demonstrates a high degree of conservation between Ginkgo and Arabidopsis.

The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba.

Ginkgotoxin (4'-O-methylpyridoxine) occurring in the seeds and leaves of Ginkgo biloba, is an antivitamin structurally related to vitamin B(6). Ingestion of ginkgotoxin triggers epileptic convulsions and other neuronal symptoms. Here we report on studies on the impact of B(6) antivitamins including ginkgotoxin on recombinant homogeneous human pyridoxal kinase (EC 2.7.1.35). It is shown that ginkgotoxin serves as an alternate substrate for this enzyme with a lower K(m) value than pyridoxal, pyridoxamine or pyridoxine. Thus, the presence of ginkgotoxin leads to temporarily reduced pyridoxal phosphate formation in vitro and possibly also in vivo. Our observations are discussed in light of Ginkgo medications used as nootropics.

The key role of peltate glandular trichomes in symbiota comprising clavicipitaceous fungi of the genus periglandula and their host plants.

Clavicipitaceous fungi producing ergot alkaloids were recently discovered to be epibiotically associated with peltate glandular trichomes of Ipomoea asarifolia and Turbina corymbosa, dicotyledonous plants of the family Convolvulaceae. Mediators of the close association between fungi and trichomes may be sesquiterpenes, main components in the volatile oil of different convolvulaceous plants. Molecular biological studies and microscopic investigations led to the observation that the trichomes do not only secrete sesquiterpenes and palmitic acid but also seem to absorb ergot alkaloids from the epibiotic fungal species of the genus Periglandula. Thus, the trichomes are likely to have a dual and key function in a metabolic dialogue between fungus and host plant.

Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia.

Individual plants belonging to different species of the family Celastraceae collected from their natural habitats in South Africa (Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl., Putterlickia pyracantha (L.) Szyszyl., Putterlickia retrospinosa van Wyk and Mostert) and Brazil (Maytenus ilicifolia Mart. ex Reiss., Maytenus evonymoides Reiss., Maytenus aquifolia Mart.) were investigated for the presence of maytansinoids and of maytansine, an ansamycin of high cytotoxic activity. Maytansinoids were not detectable in plants grown in Brazil. Analysis of plants growing in South Africa, however, showed clearly that maytansinoids were present in some individual plants but were not detectable in others. Molecular biological analysis of a Putterlickia verrucosa cell culture gave no evidence for the presence of the aminohydroxybenzoate synthase gene which is unique to the biosynthesis of aminohydroxybenzoate, a precursor of the ansamycins including maytansinoids. Moreover, this gene was not detectable in DNA extracted from the aerial parts of Putterlickia plants. In contrast, observations indicate that this gene may be present in microbes of the rhizosphere of Putterlickia plants. Our observations are discussed with respect to the possibility that the roots of Putterlickia plants may be associated with microorganisms which are responsible for the biosynthesis of maytansine or maytansinoids.

A study of the bacterial community in the root system of the maytansine containing plant Putterlickia verrucosa.

Maytansinoid compounds are ansa antibiotics occurring in the bacterium Actinosynnema pretiosum, in mosses and in higher plants such as Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl. The disjunct occurrence of maytansinoids has led to the consideration that plant-associated bacteria may be responsible for the presence of maytansinoids in P. verrucosa plants. Investigation of the bacterial community of this plant by molecular methods led to the observation that A. pretiosum, a maytansine-producing bacterium, is likely to be an inhabitant of the rhizosphere and the endorhiza of P. verrucosa.

Specificity in the interaction between an epibiotic clavicipitalean fungus and its convolvulaceous host in a fungus/plant symbiotum.

Ipomoea asarifolia and Turbina corymbosa (Convolvulaceae) are associated with epibiotic clavicipitalean fungi responsible for the presence of ergoline alkaloids in these plants. Experimentally generated plants devoid of these fungi were inoculated with different epibiotic and endophytic fungi resulting in a necrotic or commensal situation. A symbiotum of host plant and its respective fungus was best established by integration of the fungus into the morphological differentiation of the host plant. This led us to suppose that secretory glands on the leaf surface of the host plant may play an essential role in ergoline alkaloid biosynthesis which takes place in the epibiotic fungus.

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus.

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.

Clavicipitaceous fungi associated with ergoline alkaloid-containing convolvulaceae.

Ergoline alkaloids are a group of physiologically active natural products occurring in taxonomically unrelated fungal and plant taxa Clavicipitaceae (Hypocreales) and Convolvulaceae (Solanales). We show in the present paper that clavicipitaceous fungi are associated with four different ergoline alkaloid-containing plant taxa of the family Convolvulaceae. These fungi are macroscopically visible on the adaxial surface when young leaf buds are opened or are detectable by molecular biological techniques in seeds. Detectability of the fungus correlates with the absence or presence of ergoline alkaloids within the respective plant organ. The fungi contain the gene (dmaW) responsible for the committed step in ergoline alkaloid biosynthesis. Sequencing of ribosomal DNA (18S rDNA and internal transcribed spacer) as well as the dmaW gene (partial) and construction of phylogenetic trees show that the fungi are clavicipitaceous, not identical but very closely related.

Analysis of the Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis.

Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants.

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae).

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.

Polyketides from the marine-derived fungus Ascochyta salicorniae and their potential to inhibit protein phosphatases.

Chemical investigation of the marine fungus Ascochyta salicorniae led to the isolation of two new epimeric compounds, ascolactones A (1) and B (2), in addition to the structurally-related polyketides hyalopyrone (3), ascochitine (4), ascochital (5) and ascosalipyrone (6). The absolute configurations of the epimeric compounds 1 and 2 were assigned as (1R,9R) and (1S,9R), respectively, through simulation of the chiroptical properties using quantum-chemical CD calculations, and chiral GC-MS subsequent to oxidative cleavage (Baeyer-Villiger oxidation) of the side chain. In silico screening using the PASS software identified some of the A. salicorniae compounds (1-6) as potential inhibitors of protein phosphatases. Compound was found to inhibit the enzymatic activity of MPtpB with an IC(50) value of 11.5 microM.

Recent developments in the maytansinoid antitumor agents.

Maytansine and its congeners have been isolated from higher plants, mosses and from an Actinomycete, Actinosynnema pretiosum. Many of these compounds are antitumor agents of extraordinary potency, yet phase II clinical trials with maytansine proved disappointing. The chemistry and biology of maytansinoids has been reviewed repeatedly in the late 1970s and early 1980s; the present review covers new developments in this field during the last two decades. These include the use of maytansinoids as "warheads" in tumor-specific antibodies, preliminary metabolism studies, investigations of their biosynthesis at the biochemical and genetic level, and ecological issues related to the occurrence of such typical microbial metabolites in higher plants.

Bacterial production of transdihydroxycyclohexadiene carboxylates by metabolic pathway engineering.

Homochiral-cis-cyclohexa-3,5-diene-1,2-diols are important synthons. We found a way to produce trans-configured homochiral diols using recombinant Klebsiella pneumoniae 62-1. Transformation of this mutant (Phe- Trp- Tyr-) with plasmids carrying genes involved in chorismic and isochorismic acid metabolism leads to the production of either (+)-trans-(2S,3S)-2,3-dihydroxycyclohexa-4,6-dienecarboxylic acid or (-)-trans-(3R,4R)-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acid, with a yield of 70 or 90 mg (1 culture broth)-1, respectively. The metabolic shift from one diene to the other is caused by a change in activity of isochorismate hydroxymutase and/or isochorismatase which in turn results from growth under iron deficiency or overexpression of genes (entC and/or entB) involved in chrismate metabolism.

Five novel Kitasatospora species from soil: Kitasatospora arboriphila sp. nov., K. gansuensis sp. nov., K. nipponensis sp. nov., K. paranensis sp. nov. and K. terrestris sp. nov.

A polyphasic study was carried out to establish the taxonomic positions of six strains isolated from diverse soil samples and provisionally assigned to the genus Kitasatospora. The isolates were found to have chemical and morphological properties consistent with their classification as Kitasatospora strains. Direct 16S rRNA gene sequence data confirmed the taxonomic position of the strains following the generation of phylogenetic trees by using three tree-making algorithms. Five of the isolates were considered to merit species status using complementary genotypic and phenotypic data. These organisms were designated Kitasatospora arboriphila sp. nov. (HKI 0189(T)=2291-120(T)=DSM 44785(T)=NCIMB 13973(T)), Kitasatospora gansuensis sp. nov. (HKI 0314(T)=2050-015(T)=DSM 44786(T)=NCIMB 13974(T)), Kitasatospora nipponensis sp. nov. (HKI 0315(T)=2148-013(T)=DSM 44787(T)=NCIMB 13975(T)), Kitasatospora paranensis sp. nov. (HKI 0190(T)=2292-041(T)=DSM 44788(T)=NCIMB 13976(T)) and Kitasatospora terrestris sp. nov. (HKI 0186(T)=2293-012(T)=DSM 44789(T)=NCIMB 13977(T)). The remaining organism, isolate HKI 0316 (=2122-022=DSM 44790=NCIMB 13978), was considered to be a strain of Kitasatospora kifunensis on the basis of 16S rRNA gene sequence, DNA-DNA relatedness and phenotypic data.

Kitasatospora putterlickiae sp. nov., isolated from rhizosphere soil, transfer of Streptomyces kifunensis to the genus Kitasatospora as Kitasatospora kifunensis comb. nov., and emended description of Streptomyces aureofaciens Duggar 1948.

A polyphasic study was undertaken to establish the taxonomic position of a rhizosphere isolate that had been assigned provisionally to the genus Kitasatospora. The organism, isolate F18-98(T), was found to have chemical and morphological properties that were consistent with its classification as a Kitasatospora strain. Direct 16S rDNA sequence data confirmed the taxonomic position of the strain, following the generation of phylogenetic trees by using three treeing algorithms. The organism formed a 16S rDNA subclade with Kitasatospora azatica and Streptomyces kifunensis, but was distinguished readily from the latter by using a combination of biochemical and physiological properties. Genotypic and phenotypic data show that strain F18-98(T) should be classified in the genus Kitasatospora as a novel species, for which the name Kitasatospora putterlickiae sp. nov. is proposed; the type strain has been deposited in culture collections as strain F18-98(T) (=DSM 44665(T)=NCIMB 13932(T)). It is also proposed that Streptomyces kifunensis should be transferred to the genus Kitasatospora as Kitasatospora kifunensis comb. nov. An emended description of Streptomyces aureofaciens Duggar 1948 is also given.