Biosynthesis and metabolic engineering of glucosinolates

Summary. Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value. Received December 7, 2001 Accepted December 21, 2001     

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

Cytochromes P450 in gibberellin biosynthesis

The gibberellins (GAs) are an important class of plant growth regulators that are active in many aspects of plant growth and development. GAs are synthesized by a complex pathway involving three enzyme classes spanning different subcellular compartments. One of these enzyme classes is the cytochrome P450s which catalyze a number of oxidation steps in the middle part of the pathway. Mutants in these cytochrome P450-mediated steps in a number of species have been crucial in isolating the genes encoding these enzymes and have also played an important role in understanding GA physiology. GAs are also synthesized by fungi, in a biosynthesis pathway largely catalyzed by cytochrome P450s. The fungal pathway appears to have evolved independently to that of higher plants.

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Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.     

Piecing together the transport pathway of aliphatic glucosinolates

Glucosinolates are sulfur-rich secondary metabolites characteristic of the Brassicales order. Transport of glucosinolates was suggested more than 30 years ago through a number of studies which indicated that glucosinolates are produced in maternal tissue and subsequently transported to the seed. These observations laid the foundation for numerous studies on glucosinolate transport which have provided a wealth of information on biochemical properties of glucosinolate transport, source–sink relationships between organs and on the transport routes of glucosinolates. However, most of the conclusions and hypotheses proposed in these studies have not been discussed in context of each other to provide a complete overview of the current state of knowledge on glucosinolate transport. In this review, we are thus piecing together the glucosinolate pathway by presenting and critically analyzing all data on glucosinolate research. Furthermore, the data on glucosinolate transport is considered in the light of the newest findings on glucosinolate synthesis and distribution. The aim is to provide a comprehensive and updated set of hypotheses which may prove useful in directing future research on glucosinolate transport.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Polymorphic Cytochromes P450 and Drugs Used in Psychiatry

1. The cytochrome P450 monooxygenases, CYP2D6, CYP2C19, and CYP2C9, display polymorphism. CYP2D6 and CYP2C19 have been studied extensively, and despite their low abundance in the liver, they catalyze the metabolism of many drugs.2. CYP2D6 has numerous allelic variants, whereas CYP2C19 has only two. Most variants are translated into inactive, truncated protein or fail to express protein.3. CYP2C9 is expressed as the wild-type enzyme and has two variants, in each of which one amino acid residue has been replaced.4. The nucleotide base sequences of the cDNAs of the three polymorphic genes and their variants have been determined, and the proteins derived from these genes have been characterized.5. An absence of CYP2D6 and/or CYP2C19 in an individual produces a poor metabolizer (PM) of drugs that are substrates of these enzymes.6. When two drugs that are substrates for a polymorphic CYP enzyme are administered concomitantly, each will compete for that enzyme and competitively inhibit the metabolism of the other substrate. This can result in toxicity.7. Patients can be readily phenotyped or genotyped to determine their CYP2D6 or CYP2C19 enzymatic status. Poor metabolizers (PMs), extensive metabolizers (EMs), and ultrarapid metabolizers (URMs) can be identified.8. Numerous substrates and inhibitors of CYP2D6, CYP2C19, and CYP2C9 are identified.9. An individual's diet and age can influence CYP enzyme activity.10. CYP2D6 polymorphism has been associated with the risk of onset of various illnesses, including cancer, schizophrenia, Parkinson's disease, Alzheimer's disease, and epilepsy.     

Regulatory variability of camalexin biosynthesis

The anthranilate synthase ASA1, CYP79B2 and CYP71B15 (PAD3) are biosynthetic genes of the Arabidopsis phytoalexin camalexin, which are induced after pathogen infection and abiotic treatments like silver nitrate spraying. The natural variation of camalexin biosynthesis in response to Pseudomonas syringae infection was determined in several ecotypes, and differential CYP71B15 regulation as a potential basis for this variation was investigated. The expression of camalexin biosynthetic genes was restricted to the tissue undergoing cell death. After droplet infection with Alternaria alternata, a potent camalexin inducer in the Col-0 ecotype, camalexin formation and the induction of ASA1, CYP79B2 and CYP71B15 were strictly co-localized with the infection site.     

Effects of Oral Anorexiant Sibutramine on the Expression of Cytochromes P450s in Human Hepatocytes and Cancer Cell Lines

Sibutramine is a serotonin–norepine‐phrine reuptake inhibitor that was used for weight‐loss management in obese patients. Even though it was officially withdrawn from the market in 2010, it is still present in some tainted weight‐loss pills (as reported by US Food and Drug Administration). Thus, it is still reasonable to study the effects of this compound. The aim of this work was to investigate the potential of sibutramine to induce CYP1A1/CY3A4 in human cancer cell lines and CYP1A1/2, CYP2A6, CYP2B6, and CYP3A4 in human hepatocytes, a competent model of metabolically active cells. The levels of mRNA and protein of CYP1A1/1A2/3A4/2A6/2B6 were compared with the typical inducers, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and rifampicin (RIF) for CYP1A1/2 and for other CYPs, respectively. The mRNA and protein levels of all genes in either cancer cell lines or human hepatocytes were induced when treated with typical inducers but not with sibutramine.     

Breakdown of indole alkaloids in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus

The relative importance of breakdown on the accumulation of indole alkaloids has been determined in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus by the feeding of stable isotope labelled alkaloids. In all cultures a considerable amount of the alkaloid biosynthesized was broken down. The breakdown was found to be dependent on the culture period and the half-life was in the order of several days. The breakdown could not explain the difference between producing and non-producing cultures. Further it was determined that in both cultures the breakdown was due to both biotic and abiotic factors.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

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.     

Antileishmanial activity of indole alkaloids from Aspidosperma ramiflorum

The present study was designated to evaluate the antileishmanial activity of acid and basic fractions that were obtained after acid-basic extraction, from ethanolic 70% crude extract and pure compounds from the stem bark of Aspidosperma ramiflorum. The basic alkaloidal fraction presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis (LD(50) value<47 microg/ml). Based on these findings, the basic fraction was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 16.3+/-1.6 microg/ml and 4.9+/-0.9 microg/ml, respectively). Our results are promising, showing that these compounds are biologically active against Gram-positive bacteria.