Role of elements and physiologically active compounds in the regulation of synthesis and accumulation of indole alkaloids in Catharanthus roseus L

Effects of various elements (Co, Ni, Zn, W, Mn, Cr, B, Mo, Fe, and V), natural and synthetic auxins, cytokinins, and gibberellin on biosynthesis and accumulation of indole alkaloids was studied at increasing concentrations in the model system of Madagascar periwinkle seedlings (Catharanthus roseus L.). The main types of concentration dependences for the effect of physiologically active compounds under study were evaluated. A possible mechanism of the influence of Zn and auxin on this process was partly clarified. The compounds were shown to modulate various stages in the biosynthesis of monomeric indole alkaloids (catharanthine and vindoline).     

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Purification,cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family,extending the alkaloidal network of the plant <Emphasis Type="Italic">Rauvolfia</Emphasis>

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His₆-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462.     

Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus

Cell suspension cultures (cell line No 615) of Catharanthus roseus cv. Little Delicata responded to elicitor treatment by accumulating monoterpenoid indole alkaloids and phenolic compounds. The excretion of phenols into the culture medium resulted from the induction of the branch-point enzyme phenylalanine ammonia lyase. The accumulation of alkaloids, however, occurred several hours earlier than the elicitor-mediated induction of tryptophan decarboxylase through which shikimate pathway intermediates are channelled into tryptamine and related indole alkaloids. The results indicate that both pathways for phenol and indole alkaloid biosynthesis responded to elicitor treatment and that no obvious causal relationship between pathways could be deduced from this study.Abbreviations PAL phenylalanine ammonia lyase - TDC tryptophan decarboxylase Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

The Effect of Amino Acids as Components of Nutrient Medium on the Accumulation of Protoberberine Alkaloids in the Cell Culture of <Emphasis Type="Italic">Thalictrum minus</Emphasis>

We studied the effects of amino acids on the biosynthesis of protoberberine alkaloids. When 5 mM tyrosine was added to the nutrient medium, the content of alkaloids was reduced by 23% and dry weight was only 77% of the control. On the medium with 1 mM L-tryptophan, the content of alkaloids was somewhat increased (by 20%). Other amino acids (sulfur-containing L-cysteine and L-methionine, and also L-proline and L-arginine) did not affect substantially the content of alkaloids. The addition of 1 and 5 mM L-phenylalanine, which is not a primary precursor to alkaloids, induced the accumulation of alkaloids by the 17th day of the growth cycle by 40 and 140%, respectively, as compared to control treatment. The comparison of various phenylalanine concentrations showed that 7 mM phenylalanine added on the 7– 8th day induced the highest accumulation of alkaloids in the culture medium (above 1 g/l). The content of alkaloids and soluble phenolic compounds increased threefold in both the medium and cells. None of the amino acid tested enhanced biomass accumulation.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 438–442.Original Russian Text Copyright © 2005 by Urmantseva, Gaevskaya, Karyagina, Bairamashvili.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Phenolic compounds in Catharanthus roseus

Besides alkaloids Catharanthus roseus produces a wide spectrum of phenolic compounds, this includes C6C1 compounds such as 2,3-dihydoxybenzoic acid, as well as phenylpropanoids such as cinnamic acid derivatives, flavonoids and anthocyanins. The occurrence of these compounds in C. roseus is reviewed as well as their biosynthesis and the regulation of the pathways. Both types of compounds compete with the indole alkaloid biosynthesis for chorismate, an important intermediate in plant metabolism. The biosynthesis C6C1 compounds is induced by biotic elicitors.     

Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures

A callus strain with stable ability for vindoline synthesis was selected from many prepared Catharanthus roseus leaf calli to study the regulation of vindoline biosynthesis as well as other indole alkaloids. It was shown that light and plant growth regulators significantly influenced the biosynthesis of vindoline and other alkaloids as well as acidic and basic peroxidase activities. Light promoted vindoline and serpentine biosynthesis, and stimulated plastid development and peroxidase activity. However, 2,4-D suppressed the biosynthesis of all indole alkaloids and peroxidase activity. Our results suggest that light or plant hormones regulate vindoline, serpentine and other alkaloid biosynthesis and accumulation by influencing peroxidase activity and the differentiation status of callus cultures, especially chloroplast development. Some possible relationships between serpentine or vindoline biosynthesis and peroxidase activity are proposed.     

The biosynthesis and genetic engineering of bioactive indole alkaloids in plants

Indole alkaloids are widely distributed secondary metabolites that exhibit a broad range of pharmacological activities. They are synthesized through plant biosynthetic pathways involving complex enzyme activities and regulatory strategies. Since many compounds of indole alkaloids are structurally too complex to be manufactured economically by chemical synthesis, they have to be isolated from naturally grown or cultivated plants. Therefore, the biotechnological production of high-value plant secondary metabolites in cultivated cells or transgenic plants is potentially an attractive alternative. The present review describes the regulation of indole alkaloids biosynthesis, as well as their pharmacological functions in plants such as anti-microbes, anti-inflammatory and anti-tumor. Furthermore, it discusses different strategies by which the genetic engineering of indole alkaloids biosynthesis through the reconstruction of the pathway achieves high production of specific compounds.     

In‐depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites

Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D‐DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.     

Novel Indole Alkaloids from Nauclea latifolia and Their Renin‐Inhibitory Activities

Chemical studies on the crude MeOH extract of stems and barks of Nauclea latifolia resulted in the isolation of five new indole alkaloids, latifoliamides A–E ( 1 – 5 , resp.), along with one known alkaloid, angustoline ( 6 ). The structures of these compounds were elucidated by means of extensive NMR spectral studies. Compound 1 has a 20‐ethylidenetetrahydrofuran ring incorporated in its structure and represents the first example of this class of indole alkaloids. All of the isolates exhibited moderate in vitro renin inhibitory activities.     

Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy ( Papaver somniferum ), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle ( Catharanthus roseus ), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.     

Silencing of a second dimethylallyltryptophan synthase of <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> reveals a novel clavine alkaloid gene cluster

Penicillium roqueforti produces several prenylated indole alkaloids, including roquefortine C and clavine alkaloids. The first step in the biosynthesis of roquefortine C is the prenylation of tryptophan-derived dipeptides by a dimethylallyltryptophan synthase, specific for roquefortine biosynthesis (roquefortine prenyltransferase). A second dimethylallyltryptophan synthase, DmaW2, different from the roquefortine prenyltransferase, has been studied in this article. Silencing the gene encoding this second dimethylallyltryptophan synthase, dmaW2, proved that inactivation of this gene does not prevent the production of roquefortine C, but suppresses the formation of other indole alkaloids. Mass spectrometry studies have identified these compounds as isofumigaclavine A, the pathway final product and prenylated intermediates. The silencing does not affect the production of mycophenolic acid and andrastin A. A bioinformatic study of the genome of P. roqueforti revealed that DmaW2 (renamed IfgA) is a prenyltransferase involved in isofumigaclavine A biosynthesis encoded by a gene located in a six genes cluster (cluster A). A second three genes cluster (cluster B) encodes the so-called yellow enzyme and enzymes for the late steps for the conversion of festuclavine to isofumigaclavine A. The yellow enzyme contains a tyrosine-181 at its active center, as occurs in Neosartorya fumigata, but in contrast to the Clavicipitaceae fungi. A complete isofumigaclavines A and B biosynthetic pathway is proposed based on the finding of these studies on the biosynthesis of clavine alkaloids.     

<Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> associated bioactive bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.From Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 35–39.Original English Text Copyright © 2005 by Li Zheng, Xiaojun Yan, Jilin Xu, Haimin Chen, Wei Lin.This article was submitted by the authors in English.     

New insight into the biosynthesis and regulation of indole compounds in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In spite of their silent and sessile life, plants are dynamic organisms that have developed advanced defence strategies in their adaptation to the pressure of herbivores and pathogens. Natural plant products play an important role as chemical weapons in this warfare. Characteristic of cruciferous plants is the synthesis of nitrogen- and sulphur-rich compounds, such as glucosinolates (Mikkelsen et al. 2002) and indole alkaloids (Pedras et al. 2000). Glucosinolates are believed to be largely non-toxic, but upon tissue disruption, they are hydrolyzed by endogenous -thioglucosidases (myrosinases) (Rask et al. 2000) to primarily isothiocyanates and nitriles, which have many biological activities. These include not only important roles as repellents against herbivorous insects and microorganisms, but also as volatile attraction of specialized insects (Wittstock and Halkier 2002). For humans, these compounds serve as cancer-preventive agents, biopesticides, and flavor compounds (Talalay and Fahey 2001). Indole alkaloids are phytoalexins and production of specific alkaloids is usually limited to only a few species. Cruciferous plants include the model plant Arabidopsis, which produces the indole alkaloid camalexin. This review will focus on the central role of indole-3-acetaldoxime (IAOx) in the biosynthesis of indole glucosinolates, camalexin, and the phytohormone IAA.     

Rare Bisindole Alkaloids from the Amazonian Tree Raputia simulans

The stem bark of Raputia simulans (Rutaceae) has been reported to contain simple and dimeric indole alkaloids. Further phytochemical investigation of R. simulans stem bark resulted in the isolation of three new alkaloids. These compounds represent a relatively new category of dimeric indole alkaloids with a cyclohexene moiety in their core. Their structure elucidations were based on NMR and HR‐MS techniques, while structural aspects concerning their relative configuration were investigated using molecular mechanics calculations and NOESY experiments.     

Violacein and related tryptophan metabolites produced by <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis>: biosynthetic mechanism and pathway for construction of violacein core

Violacein is a natural violet pigment produced by several Gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH₂ at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006–2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.     

Differentiation and melanin production in hyaline and pigmented strains of Microdochium bolleyi

The effect of various compounds on growth, melanin biosynthesis and cell differentiation was studied in a hyaline (SH25) and a pigmented (SH25B) strain of Microdochium bolleyi. Dark pigment production by the hyaline strain was induced by the presence of DOPA and indole in the medium, both of which are intermediates in the tyrosine pathway of melanin biosynthesis. These substrates also induced conidia and chlamydospore production, structures which were not normally seen in the hyaline strain. The systemic fungicide, tricyclazole, inhibited melanin synthesis in the pigmented strain but not the development of chlamydospore-like cell structures.