The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).     

Pubescine A,a cyclopeptide alkaloid from Discaria pubescens

From Discaria pubescens, a new peptide alkaloid, pubescine A (1), belonging to the frangulanine type has been isolated and its structure elucidated. Pubescine A seems to be isomeric to melonovine A. For the first time, d-leucine has been identified in a cyclopeptide alkaloid.     

A new peptide alkaloid from Discaria crenata

The structure of a new peptide alkaloid, crenatine A isolated from Discaria crenata, has been elucidated.     

Isolation and purification of peptide and alkaloid toxins from nabaena flos-aquae using high-performance thin-layer chromatography

A procedure has been developed for the sequential extraction and purification of the peptide and alkaloid toxins from a single batch of Anabaena flos-aquae cells. After solvent extraction and adsorption onto C₁₈ Sep-Pak cartridges, the cyanobacterial toxins were purified by high-performance thin-layer chromatography and identified by scanning with a densitometer. The purified components were tested separately by mouse bioassay. Liver damage and nervous disorder were induced by the two peptide and single alkaloid toxins, respectively.     

Peptide alkaloids of Scutia buxifolia

Two new components, the peptide alkaloid scutianine D and scutianene C have been isolated from Scutia buxifolia and their structures elucidated. The configuration of some of the asymmetric centers of scutianine A has been determined by gas chromatography.     

Peptide and tetrahydroisoquinoline alkaloids from Euonymus europaeus

Three p-aryloxy macrocyclic peptide alkaloids frangulanine, franganine and frangufoline together with the 1-benzyltetrahydroisoquinoline alkaloid armepavine have been isolated from Euonymus europaeus native to Poland. These types of alkaloids are new to the Celastraceae and are of chemotaxonomic interest.     

Sativanine-g,a cyclopeptide alkaloid from Zizyphus sativa

In addition to the already described peptide alkaloids from the bark of Zizyphus sativa, a new compound of this class, sativanine-G, has been isolated and its structure elucidated. This alkaloid contains a 13-membered ring system and belongs to the nummularine-C class.     

Cyclopeptide alkaloids from Zizyphus nummularia

In addition to the known alkaloid nummularine-B, two new peptide alkaloids nummularine-M and nummularine-N have been isolated from Zizyphus nummularia and their structures elucidated. Nummularine-M is a 14-membered cyclopeptide and belongs to the integerrinine type, whereas nummularine-N is a 13-membered cyclopeptide like nummularine-B.     

Scutianin-G,ein weiteres cyclopeptidalkaloid aus Scutia buxifolia

From the bark of Scutia buxifolia, in addition to already described peptide alkaloids, a new compound of this class, Scutianine-G, has been isolated and its structure elucidated. This alkaloid contains a 14-membered ring system and belongs to the Frangulanine-type; it is diastereoisomer of Scutianine-D and -E.     

Na-formylechitamidine,an alkaloid from Alstonia boonei

A known alkaloid, echitamidine, and a new alkaloid, N_a-formylechitamidine, have been isolated from the stem bark of Alstonia boonei. The structure of this new alkaloid was assigned on the basis of chemical and spectroscopic data.     

Some alkaloids of Alstonia undulata

Sixteen indole alkaloids have been identified in the root bark and in the leaves of Alstonia undulata from New Caledonia. They are vincamedine, tetrahydroalstonine, alstonidine, deplancheine, fluorocarpamine, pleiocarpamine, 11-methoxyakuammicine, cabucraline, desoxycabufiline, cabucraline-N(4)-oxide, 11-methoxyakuammicine N(4)-oxide, gentiacraline, plumocraline, vincorine, cathafoline and pericyclivine. Five bis indoles of unknown structure have been isolated. The root bark alkaloid mixture contains gentiacraline the first alkaloid made up of an indole and a pyridine alkaloid.     

Heterologous expression of alkaloid biosynthetic genes — a review

Tetrahydrobenzylisoquinoline alkaloids comprise a diverse class of secondary metabolites with many pharmacologically active members. The biosynthesis at the enzyme level of at least two tetrahydrobenzylisoquinoline alkaloids, the benzophenanthridine alkaloid sanguinarine in the California poppy, Eschscholtzia californica, and the bisbenzylisoquinoline alkaloid berbamunine in barberry, Berberis stolonifera, has been elucidated in detail starting from the aromatic amino acid (aa) l-tyrosine. In an initial attempt to develop alternate systems for the production of medicinally important alkaloids, one enzyme from each pathway (BBE, a covalently flavinylated enzyme of benzophenanthridine alkaloid biosynthesis and CYP80, a phenol coupling cytochrome P-450-dependent oxidase of bisbenzylisoquinoline alkaloid biosynthesis) has been purified to homogeneity, a partial aa sequence determined, and the corresponding cDNAs isolated with aid of synthetic oligos based on the aa sequences. The recombinant enzymes were actively expressed in Spodoptera frugiperda Sf9 cells using a baculovirus vector, purified and then characterized. Insect cell culture has proven to be a powerful system for the overexpression of alkaloid biosynthetic genes.     

An N-formyl cyclopeptide alkaloid from the bark of zizyphus sativa

From the bark of Zizyphus sativa, previously undescribed cyclopeptide alkaloid, sativanine-F has been isolated. The structure was deduced by spectroscopic methods and chemical degradation. It is a 13-membered cyclopeptide alkaloid and provides the first example of such a naturally occurring N-formyl cyclopeptide alkaloid.     

Indole alkaloids from Alstonia scholaris

A new indole alkaloid, alstonamine and a sitsirikine type indole alkaloid, rhazimanine, have been isolated from the leaves of Alstonia scholaris.     

N-isobutyl-trans-2-trans-4-eicosadienamide and other alkaloids of fruits of Piper guineense

A novel alkaloid, N-isobutyl-trans-2-trans-4-eicosadienamide, has been isolated from the fruits of Piper guineense and fully characterized. The structure of the compound has been confirmed by an unambiguous synthesis of the tetrahydro derivative. The known alkaloid, δαβ-dihydropiperine has also been isolated and the position of the double bond in this compound confirmed by the use of an NMR shift reagent.     

Structure revision of emiline a pyrrolizidine alkaloid from Emilia flammea

The structure of emiline, a pyrrolizidine alkaloid isolated fromEmilia flammea has been revised from an 11-membered alkaloid to a 12-membered macrocyclic diester containing otonecine.     

Enzymatic synthesis of cyclopeptine intermediates in Penicillium cyclopium

Cyclopeptine, a benzodiazepine alkaloid of Penicillium cyclopium, is formed from anthranilic acid, L-phenylalanine and the methyl group of L-methionine by cyclopeptine synthetase. The following partial activities of this enzyme system were determined in vitro: anthranilic acid and L-phenylalanine adenylyltransferase activity, binding of anthranilic acid and L-phenylalanine as thioesters to proteins, formation of thioester-bound N-methyl-L-phenylalanine and N-methyl-L-phenylalanylanthranilic acid. The obtained results indicate that cyclopeptine is formed via enzyme-bound intermediates by the thiotemplate mechanism of peptide biosynthesis.     

Pachyovatamine,a bisbenzylisoquinoline alkaloid,and other alkaloids from Pachygone ovata

A new dibenzo-p-dioxin biphenyl bisbenzylisoquinoline alkaloid, pachyovatamine, has been isolated from an extract of the leaves and stems of Pachygone ovata from Sri Lanka. The alkaloid was characterized by a consideration of its physicochemical data and conversion to O-acetyltiliacorinine. Pachygonamine, N-methylpachygonamine and tiliamosine were also isolated from the same extract.     

Structure of erysophorine: a new quaternary alkaloid of Erythrina arborescens

Isolation and structure elucidation of a new quaternary indole-eryso alkaloid, named erysophorine, from seeds of Erythrina arborescens are reported. An indole-erythrina alkaloid, such as erysophorine, has not been encountered before in nature or prepared synthetically.