Genetic approach to alkaloids

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Alkaloid biogenesis in an organism is determined gene-tically16'526'527'528'529'530'531'532'533'534'535. This means that many specific genes participate in alkaloid metabolism, and gene participation in metabolism is a very important basis for understanding the alkaloids. As is widely recognized, the gene is a unit of hereditary information encoded in a discrete segment of a DNA molecule, which carries an enormous amount of genetic information. It has been generally estimated that human cells contain from 50000 to 100000 genes on 23 chromosomes. The initial results of the Human Genome Project have been published beginning in June 2000 and finally in 2003. As one result of the project, it became clear that the human genome has only 30000-40000 genes, which was less that expected in previous estimations536'537. The mouse (Mus musculus) has about 25 000 genes, the nematode (Caenorhabditis elegans) 19000 genes, the fruit fly (Drosophila melanogaster) about 13 700 genes and the common wall cress plant (Arabidopsis thaliana) has 25 500 genes538. Genetic information connecting to the metabolism of alkaloids signifies that these secondary compounds are more important for the life cycle of organisms as they are not coded in the genome. Lal and Sharma539 have studied alkaloid genetics in P. somniferum. The alkaloids of this plant are determined by dominant and recessive genes. The inheritance of morphine, codeine and thebaine content from parent plants to the next generation is 21-36%, and that of narcotine only 10.5-14.5%539. The authors539 have also cited previous work of Briza, according to which the heritability of morphine content in P. somniferum ranged from 43% to 68%. When considering narcotine content, some degree of epistasis is reflected539. The dominant and recessive gene determination of alkaloid content makes alkaloid genetics a very difficult research topic. However, to date more than 30 genes coding for enzymes involved in alkaloid biosynthesis pathways have been isolated and cloned (Table 22). Recently, acetylajmalan esterase (AAE) was isolated and purified together with a full-length AAE cDNA clone from Rauvolfia cells534. This enzyme plays an essential role in the late stages of ajmaline biosynthesis. This was the eighth functional alkaloid gene extracted from this plant. This was also the sixth identified

Table 22 Enzymes specifically involved in alkaloid biosynthesis

Alkaloids of Plant Species

Enzymes

Coded by DNA

Purine alkaloids

Pyrrolizidine alkaloids

Indole alkaloids

Isoquinoline alkaloids

Tropane alkaloids

Caffeine synthase Xanthosine 7-N-methyltransferase 7-Methylxanthine 3-N-methyltransferase Caffeine xanthinemethyltransferase

1 (CaXMTl)

Caffeine methylxanthinemethyltransferase

2 (CaMXMT2) Caffeine

Dimethylxanthinemethyltransferase (CaDXMTl) Theobromine 1-N-methyltransferase

Homospermidine synthase

Tryptophane decarboxylase

Secologanin synthase Strictosidine synthase

Polyneuridine aldehyde esterase Taberosine 16-hydrolase Desacetoxyivindoline acetyltransferase Geraniol/nerol 10-hydroxylase

Tyrosine/DOPA decarboxylase

Berberine bridge enzyme Norcoclaurine 6- O-methyltransferase Coclaurine N-methyltransferase 3'-Hydroxy-N-methylcoclaurine

4- O-methyltransferase Scoulerine 9-O-methyltransferase Columbamine O-methyltransferase O-methyltransferases N-methylcoclaurine 3'-hydroxylase Berbamunine synthase Codeinone reductase Salutaridinol 7-O-acetyltransferase

Hyoscyamine 6j6-hydroxylase Tropinone reductase-I Tropinone reductase-II

Camellia sinensis, Coffea arabica Coffea arabica Coffea arabica Coffea arabica

Coffea arabica

Coffea arabica

Coffea arabica

Senecio vernalis, Senecio vulgaris

Catharanthus roseus Camptotheca acuminata Catharanthus roseus Catharanthus roseus Rauvolfia serpentina Rauvolfia serpentina Catharanthus roseus Catharanthus roseus Catharanthus roseus

Papaver somniferum Arabidopsis thaliana Eschscholtzia californica Coptis japonica Coptis japonica Coptis japonica

Coptis japonica Coptis japonica Thalictrum tuberosum Eschscholzia californica Berberis stolonifera Papaver somniferum Papaver somniferum

Hyoscyamus niger Atropa belladonna Hyoscyamius niger Datura stramonium Hyoscyamius niger Datura stramonium Solanum tuberosum

Acridone alkaloids

Acridone synthase

Ruta graveolens

Sources: Refs [527, 532, 54l, 542, 543, 544, 546, 547, 548, 549, 550, 55l, 552, 553, 554, 555, 556, 557, 558, 559, 560, 56l, 562, 563].

ajmaline biosynthetic pathway-specific gene. Strictosidine synthase with cDNA and Genomic DNA (strl) has been isolated from Rauwolfia serpentina and Rauwolfia mannii527'540'541. This enzyme coded in cDNA has been isolated also from Catharanthus reseus542'543. Tryptophan decarbocylase endoded by cDNA has also been isolated from this plant and then described544. Moreover, Cane et al.533 recently discovered that the synthesis of the nicotine in the roots of Nicotiana tabacum is strongly influenced by the presence of two non-allelic genes, A and B.

Hibi et al.545 have reported on putrescine N-methyltransferase isolated from the nicotine biosynthetic pathway coded by cDNA. Recent advances in cell and molecular biology of alkaloid biosynthesis have heightened awareness of the genetic importance.

Biosynthetic genes involved in the formation of tropane, benzylisoquinoline and terepenoid indole alkaloids have been isolated561. Hyoscyamine 6-^-hydrolase563 and tropinione reductases548 encoded in cDNA and involved with tropane alkaloids have been isolated as well. Moreover, some genes involved in the metabolism of isoquinoline alkaloids and encoded in cDNA are also known. The berberine bridge enzyme from Eschscholtzia californica541 and berbamurine synthase from Berberis stolonifera551 belonging to this group of enzymes are encoded in cDNA. Coclaurine N-methyltransferase and columamine O-methyltransferase involved in the biosynthetic pathways of the isoquino-line alkaloids, berberine and palmatine respectively have been found and cloned in Coptis japonica553'554. Salutaridinol 7-O-acetyltransferase forms an immediate precursor of thebaine along the morphine biosynthetic pathway, and its cDNA was obtained from a cell suspension culture of the opium poppy (P. somniferum)553'554. Caffeine is formed from xanthosine through three successive transfers of methyl groups and a single robose removal in coffee plants. The methylation is catalysed by three N-methyltransferases: xanthosine methyltranserase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethyltransferase (DXMT), which participates in the caffeine synthetic pathway552.

There is the evidence that genes involved in alkaloid metabolism can be isolated and engineered to new plants. The biotechnological potential is apparent especially in cytochrome P450 genes isolated from Catharanthus roseus. P450 genes involve in the 16-hydroxylation of tabersonin in this plant and establish recombinant system CY71D12 as a tabersonine 16-hydroxylase. In Lonicera japonica P450 genes involve in the conversion of loganin into secologanin systems as CYP72A1 as a secologanin synthase and CYP76B6 as geraniol/nerol 10-hydroxylase. CYP72A1 from higher plants catalyse ring-opening reactions. CYP76B6 and CYP71D12 catalyse alkaloid moiety. In the indole alkaloid biogenesis P450 genes catalyse a large of number of reactions, for example P450 genes are important in the formation of parent ring systems of alkaloids. Engineered plant defence and herbicide tolerance is developed by transferring of some 450 genes. Animal enzymes encoded by P450 indicate potential use in plant defence system after their translocation by biotechnological engineering557. Knowledge of these key genes can be used to enhance alkaloid production in the cell cultures557 562. The biological importance of alkaloids is connected with the structural, metabolic, functional and evolutionary role of these compounds in living organisms. The present research on the genes involved in the biosynthesis of alkaloids is advanced and many enzymes have been isolated and cloned. However, the major challenge in the near future is to isolate new genes and new enzymes. Research needs to uncover more information about the regulation of metabolism at different levels, such as genes, enzymes, alkaloid production and accumulation. A challenge in this research area will be to provide more data on genetic information as a means of mapping metabolic networks between these levels. This could help develop better models for alkaloid biosynthesis and production, which will support the metabolic engineering of alkaloids in the future.

7. Alkaloids in the evolution of organisms

Alkaloids hold many secrets of life. They are toxic and many of them can be used as narcotics. As important secondary compounds, they categorically determine much about life. They also play a large role in evolution due to their characteristics.

Charles Darwin's theory of evolution revolutionized biology and has motivated biologists to make empirical studies of evolutionary phenomena in nature and in the laboratory. As a result of this, a fundamental science presently exists based on this theory. A serious biologist must pay heed to Darwin's statements along with later neo-Darwinistic developments and Mendelism when searching for a deeper understanding of life. Molecular biology, which is presently powering all the biological sciences, is strengthened by Darwin's and Mendel's theories and has completely supported them.

There are many recent studies that consider evolution and co-evolutionary interactions between plants and insects564'565'566'567'568'569. Many of these proved that there is interdependence between plant chemistry and the animals which feed on these plants, especially with insects570'571'572'573'574'575'576'577'578. Literature is accordant in pointing out the importance of plant and animal chemistry in both evolutionary and co-evolutionary processes. Alkaloids are good examples of this chemical role. The classical example is the potato beetle Lep-tinotarsa decemlineata living on the S. tuberosum and other Solanum species. These species contain solanine, solanidine and other minor steroid alkaloids. Solanine and solanidine are toxic. However, L. decemlineata tolerates these alkaloids when feeding (on the green mass of potato). Moreover, L. decemlin-eata does not store these alkaloids in its body and they are eliminated during metabolism. The study concerning the effects of quinolizidine alkaloids on the potato beetle (Leptinotarsa decemlieata) proved that these alkaloids reduce populations of Leptinotarsa and the development of their larvae232. Elsewhere, in a case concerning steroid alkaloids (solanine, solanidine), the Colorado beetle has not adapted to the alkaloid lupin. Moreover, in co-evolutionary development some aphids not only feed on alkaloid plants, but also sequester the alkaloids and keep them in the own body. Examples of this are the case of Macrosi-phum albifrons with quinolizidine alkaloids or the case of Aphis jacobaeae or ladybirds (Coccinella) with pyrrolizidine alkaloids. On the other hand, it is necessary to pay attention to the fact that aphids, lady birds and other insects are feeding on the alkaloid poor, or alkaloid-free forms of the same species. This can be explained as some example of co-evolutionary development. Alkaloids are molecules developed in co-evolutionary processes with environment. The evolution of the ability to use some alkaloids by some insects is a consequence of this (Figure 91). When food sources change, an organism needs to adapt to the new conditions. This is a basic matter of evolution. Moreover, cytochrome c, one of the basic enzymes, exists largely in plants, animals, fungi and some bacteria as, for example, Rhodospirillum rubrum. Nearly 60% of amino acids of cytochrome c in the homogenic position are identical in wheat and human and even 30% are identical in R. rubrum and human. This is only one piece of evidence that genes for cytochrome c have evolved from the first gene of

Changes

Changes

BEHAVIOUR

TOXICITY TOLERANCE

Changes

BEHAVIOUR

TOXICITY TOLERANCE

Changes pre-bacteria in the history of life on the Globe. Genes for alkaloids should be also evolved in a similar way.

New evidence of evolution is presently available. Evolution of viruses as complex genomes564 and the development of nucleoprotein into RNA and after that to DNA are two hypotheses considered very important for understanding the mechanisms of life565. It has been stated that alkaloids can influence DNA and RNA as well as protein synthesis in general because their metabolism is encoded genetically. Even the smallest changes in the gene code influence this mechanism. The starting point for all changes is the cell.

The chemical behaviour of one organism is affected that in another. Jackson et al.566 have described the evolution of anti-predator traits in response to a strategy by predators, and Lion et al.579 addressed the evolution of parasite manipulation of host dispersal behaviour. This study reveals that parasites can manipulate their host's dispersal. The evolution of herbivore-host plant specialization requires low levels of gene flow between populations. Leonardo and Mondor567 showed that the facultative bacterial symbiont Candidatus regiella insecticola alters both dispersion and mating in the pea aphid Acyrthosiphon pisum. Changes in dispersal and mating associated with symbionts are likely to play a key role in the initiation of genetic differentiation and in the evolution of pea aphid-host plant specialization.

The evidence of the participation of alkaloids in the evolution of organisms is observed in interactions with numerous micro-organisms. As it has been stated, many alkaloids have antimicrobial activity. However, there are several alkaloids without this characteristic. Some micro-organisms as symbionts of Bradyrhizobium spp. can live in both alkaloid-rich and alkaloid-poor plants with the some level of activity. The same can be stated in connection to some fungi, for example mycorhiza. Adaptation processes in nature lead to permanent evolution and co-evolution between alkaloids as a part of biochemistry and organisms (as a part of environment). The evolution and ongoing co-evolution of alkaloids and organisms is an example that alkaloidal defence of a plant is only a secondary function of these molecules that also changes in the evolutionary process.

CHAPTER 4

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