Biogenesis of alkaloids

The synthesis and structural analysis of alkaloids leads to the following basic questions: why are alkaloids synthesized in an organism and on which mechanism is alkaloid formation and degradation dependent in the life cycle? It is known that alkaloids have a genetic nature59 and that alkaloid content is diverse inside and between the species16. In nature the same species of plants may have both high and low alkaloid content120121. Natural hybridization has been successfully used in plant breeding for the development of the so-called "sweet cultivars" in crop production. "Sweet cultivars", however, are not without alkaloids. The total removal of alkaloids is impossible. "Sweet cultivars" are therefore plants, in useful organs of which alkaloids are present at a very low level, the bioac-tivity of which is not of any significant or observable level. However, alkaloid decrease by hybridization is an undirect but strong argument for the case that alkaloids have an heredity nature and that their presence in plants is of an evolutional character. This is fundamental in answering the first question connected with the biogenesis of alkaloids. Alkaloids have a strong genetic-physiological function and background in the organisms which produce them. The biogenesis of alkaloids is therefore a part of the total genetic-functional strategy of such metabolisms.

2.8.1. Chemistry models

From the year 1805, when alkaloid chemical research started, the problem of the biogenesis of alkaloids proved central for chemists. The background to this problem was the fact that chemical compounds are synthesized by plants, used by plants and degradaded by plants. In the case of alkaloids, it was still difficult in the middle of the 20th century to truly ascertain the purpose of alkaloids in plants. Certainly, the use of these compounds in many applications outside of the organisms producing them was well recognized. Their role within the plants, especially in the metabolism, was not known. The general consensus was that alkaloids were "the waste" product of metabolisms and had no active role to play16. Therefore, chemical chains of alkaloid production were explained as chemical reactions, the "technical" process of life. Later, especially from the late 70s of the 20th century, the theory of "wastes" was debated and corrected16. However, chemical research has now extensively proved the existence of new alkaloids, the pathways of their biosynthesis and structural modification. Three directions in this research have been followed, one purely chemical, the second, biochemical, and the third purely biomolecular, or the molbiological direction.

The chemical explanation of alkaloid biogenesis is based on the consideration that all reactions are of a chemical nature and that the energy needed for life is produced by chemical reactions. Figure 73 shows a diagram of the chemical explanations for alkaloid biogenesis. From this diagram, it is clear that alkaloid

Precursor

Obligatory O_

intermedia

Second obligatory ntermedia c

Postcursors 1-i-

Organisms producing alkaloids >

ALKALOIDS

Functional chemical receptors in metabolism system

Chemical degradation c c

Primary metabolism

Secondary metabolism blocks

Synthesis pathways

Figure 73. Chemical explanation for alkaloid biogenesis in organisms (c = catalysers).

is one of the metabolic objects in the system. It has a long chemical chain, which includes chemical synthesis before and chemical degradation after its functional activity in the metabolism. Biogenesis is, therefore, considered by chemistry to be the chain of the reactions between chemical molecules and by chemical means, in which reactions, conditions and catalysers are of special importance. Chemistry and organic chemistry consider alkaloid biogenesis to be the transformation of organic material with reaction catalysers. Different alkaloids have their own biogenesis and they are used, separately or together, with biochemical models in developing the methods for synthetic reactions and the modification of structures. Moreover, these models are also used in biotechnology263 264. Figure 74 presents the chemical model for the synthesis of Catharanthus alkaloids. It shows the primary metabolism as the background for alkaloid formation, although the Catharanthus alkaloids are the yields of a secondary metabolism. The connection between primary and secondary metabolisms is an important area for future studies in chemistry. From the model presented, it is clear that Catharanthus alkaloids are postcursors from three basic compounds: acetate, glucose and tryptophan. In the Catharanthus alkaloids, three types of ring nucleus are presented. The chemical model describes biogenesis from the point of view of the formation nucleus and skeleton of alkaloids, together with connected chemical molecule reactions in their structural and dynamic changes. Torssell225 has used the term "mechanistic approach" for the secondary metabolism to describe the chemical approach

Giao Mam Non
Figure 74. Chemical model of indole alkaloid formation in Catharanthus roseus. The arrows represent the direction of formation, the flux of compounds skeleton construction.

to this metabolism. Chemical routes, and alternative routes and their options, are very important for chemical models. In particular, the tendency of natural processes and reactions to shorten synthesis pathways is significant. Nowadays, the principles of alkaloid biochemistry and their biosynthetic means are widely recognized by specialists, and chemical, or mechanistic approaches to synthesis and biosynthesis, from a basic part of research. Without carbons, nucleus, skeleton, ring and moiote, the alkaloid will not exist. To research these structural components of alkaloids, chemical models of approach are the most effective.

2.8.2. Biochemistry models

The description of single enzyme activity in chemical reactions, together with the activity of other biomolecules, is typical for biochemical models of alkaloid biogenesis. There is no contradiction between chemical and biochemical, which serve to enrich one another. In many cases, typical chemical and biochemical models are unified in papers today263 264.

Biochemical reactions are basically the same as other chemical organic reactions with their thermodynamic and mechanistic characteristics, but they have the enzyme stage. Laws of thermodynamics, standard energy status and standard free energy change, reduction-oxidation (redox) and electrochemical potential equations are applicable to these reactions. Enzymes catalyse reactions and induce them to be much faster225 265. Enzymes are classified by international convention into six classes on the basis of the chemical reaction which they catalyse. According to Enzyme Commission (EC) rules, the enzyme classes are (1) oxidoreductases (transfer of hydrogen or oxygen atoms and electron forms), (2) transferases (transfer of chemical groups), (3) hydrolases (catalysing of hydrolytic reactions), (4) lyases (cleaving substrates by other reactions than hydrolysis, (5) isomerases (intramolecular rearragements), (6) synthases (catalyse covalent bond formation). The best-known enzymes and coenzymes active in alkaloid biogenesis are presented in Table 20.

The biochemical model contains the pathways of the enzymatic reactions in the synthetic routes. Model can be constructed for each alkaloid. Figure 75 presents biochemistry model of Catharantus alkaloids. The most important enzymes on this model are TDC (tryptophan decarbocylase), G10H (geraniol 10-hydroxylase) and SS (strictoside synthase). NADPH+, PO (Peroxidase), O (oxidase) and NADH+ are all active in different Catharantus alkaloid formations. The biochemical models are subject to both qualitative and quantitative alkaloid

Table 20 Some well-known enzymes and coenzymes active in alkaloid biogenesis

Enzyme Type

Reactions

Decarboxylases (DC)

Tryptophan decarboxylase (TDC) Phenylalanine decarboxylase (PDC) Dimerases (DM) Hydroxylases (H) Methylases (MT) Synthases Oxidases (O) Peroxidases (PO) N-methyltransferase (MT) Amine oxidases (AO)

Monoamine oxidase (MO) Diamine oxidase (DO) Dehydrogenases (DHG)

NAD+(nicotinamide adenine dinucleotide) NADP+(nicotinamide adenine dinucleotide phosphate) Pyridoxal phosphate (PLP)

S-adenosylmetionine (SAM)

Dimethylallyl diphosphate (DMAPP) Transaminases (TA) Reductases (RD)

Decarboxylation

Dimerisation Hydroxylation +CH3 Synthesis

Removing hydrogen from a substrate Using hydrogen peroxide Transfer of methyl group Oxidizing reactions Dehydrogenation to an imine Oxidizing to aldehyde Removes two hydrogen atoms from the substrate

Tends to be utilized as hydrogen acceptor Tends to be utilized as hydrogen acceptor

Coenzyme in transamination and decarboxylation Involves biological reactions Provides positively charged sulphur and facilitates nucleophilic substitution Nucleophilic substitution Transamination Reduction

Sources: Refs [32, 308, 529, 553, 639, 677, 769, 770, 771, 772, 773, 774, 775].

Biogenesis AlkaloidsBiogenesis Alkaloids
Figure 75. The biochemical model for indole alkaloid formation in Catharanthus roseus. The arrows represent the direction of the formation and the flux of compounds in skeleton construction. On the diagram, enzymes are shown by a circle.

analysis. Not all enzymes participating in alkaloid synthesis and degradation are yet known. Alkaloid enzymatology is, therefore, a growing research area.

2.8.3. Molecular biology models

Alkaloid research and bioanalysis of central-processing molecules (DNA and RNA) led to the important concept of the heredity nature of alkaloid metabolisms. Recent investigations have proved empirically that alkaloids have a genetic background and that all their biogenesis is genetically determined266'267'268'269'311. According to Tudzynski et al.266, cpdl gene coding for dimethylallyltryptophan syntase (DMATS) catalyses the first step in the biosynthesis of ergot alkaloids from Claviceps purpurea. The second gene for ergot alkaloid biosynthesis is cppsl, which encodes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. According to Tudzynski and his research group266, this protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide

(17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthase 1 (LPS1) of C. purpurea. The authors proved that cpps1 encodes LPS1. Cpd 1 is also involved in ergot alkaloid biogenesis. Cpox 1 probably encodes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), while the second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation266.

At least some genes of ergot alkaloid biogenesis in C. purpurea were found to be clustered. This means that detailed molecular genetic analysis of the alkaloid pathway is possible266. These results were confirmed by the research of Haarmann et al.269. Moreover, Huang and Kutchan267 found three genes (cyp80b1, bbe1 and cor1) which encode the enzymes needed for sanguinarine synthesis. Molecular biology models may be constructed for each alkaloid biogenesis. An example of this kind of model is presented in Figure 76.

Molecular biology research on alkaloids is very revealing. Its results can be used in the construction of alkaloid biogenetic models. At present, only a few alkaloid metabolism genes are known.

2.8.4. Analytical dilemmas

Chemical, biochemical and biological models of alkaloid biogenesis can only be constructed according to scientific research on the small chains of the synthesis of each alkaloid, and enzyme and gene involved in these chains. Models are constructed from the experimental data on synthesis and degradation of alkaloids.

Figure 76. Molecular biology model of Claviceps purpurea alkaloids.

Despite the fact that a high technical level of analytical equipment exists and research is exact, the results do not give a direct answer to some questions of biogenesis. For structural (chemical), enzymatic (biochemical) and genetic (molbiology) research, different techniques are deployed. They work in different conditions and the results received are from these different conditions, although from the same places in the pathway. In model construction, a researcher should unify these results for a logical chain. In many cases, there are theoretical or hypothetical conclusions. Certainly, the common analysis in the same trial of the structural, biochemical and biological aspects of alkaloids will be the best method. Such kinds of supertechnique are still not developed. Therefore, the primary analytical dilemma concerns the question of separate or common analysis4. On the one hand, this question covers the chemical, biochemical and molbiology aspects and, on the other, the isolation of alkaloids. The dominant way is separate analysis for separate alkaloids. Separate alkaloid analysis gives exact results in microscale, microplace and microimportance. In many cases, such results would be compromised in macroscale analysis (the biogenesis model). However, in the extraction of some group of alkaloids, the same method (common in this sense) is accepted in many papers. One example is quinolizidine alkaloids. Although quinolizidine alkaloids are very similar, they also have differences in optimal dissolving (temperature and pH-value), purity, stability and (+)- and (—)-forms. The same method of extraction for all alkaloids is a great compromise and causes compromised results, which have been regarded as sufficient.

Another analytical dilemma is the problem of in vitro and in vivo conditions. Alkaloids should be studied in their physiological conditions in organisms. This is not possible in many cases. In vitro experiments give compromised data. Theoretical conclusions and hypotheses in analysis, although they are in many cases indicators of a new breakthrough, also have some problems and some risks.

Analytical dilemmas are one reason why continuous novelization of structural, biochemical and molbiological results is necessary. These dilemmas merit attention nowadays, more than 200 years after the first alkaloid was isolated.

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