Alkaloid Formation

Fig. 10.1 Reaction catalyzed by strictosidine synthase (Str) in monoterpenoid indole alkaloid formation.

Since 1988, the methods that we use to isolate cDNAs of alkaloid biosynthesis have become ever more facile and sensitive, allowing for more efficient cDNA identification. We do not, however, yet understand enough about the cellular localization of alkaloid formation or about the nature of the catalysts to move completely away from enzymology and biochemistry and to use only molecular genetic techniques to dissect these biosynthetic pathways. Even our most recently successful cDNA isolations and identifications involved classical protein purification. We are beginning now to use proteomics and EST sequencing to identify natural product biosynthetic cDNAs, but these approaches are more feasible when a specialized cell/tissue type in which secondary metabolite biosynthetic pathways are active, can be isolated and used as a protein or RNA source. 

Frischknecht, P. M., Ulmer-Dufek, J., and Baumann, T. W. (1986). Purine alkaloid formation in buds and developing leaflets of Coffea arabica expression of an optimal defence strategy. Phytochemistry 25,613-616. 

Site of alkaloid formation, transport, and accumulation. QA are formed in the aerial green parts of legumes, especially in the leaves (.9) In lupin leaves we succeeded in localizing the key enzymes of QA biosynthesis in the chloroplast (10, 11), where the formation of the precursor lysine also takes place. Like most of the processes that are located in the chloroplast, QA biosynthesis is regulated by light (.8) and QA formation fol lows a light-dependent diurnal rhythm (, 13). The alkaloids formed in the leaves are translocated via the phloem (13, 14) all over a lupin plant, so that all plant parts contain alkaloids. QA are accumulated and stored preferentially in epidermal and subepidermal tissues of stems and leaves (15, 16). Especially rich in alkaloids are the seeds, which may contain up to 5% (dry weight) alkaloid (equivalent to 200 mmol/ kg). ... 

Natural sesquiterpene pyridine alkaloid formation needs two precursors, one for the pyridinium moiety and another for the sesquiterpene moiety. The a for formation of the pyridinium moiety is nicotinic acid, which reacts with isoleucine and, by oxidative reaction, produces evoninic acid, wilfordic acid or edulinic acids, a for the sesquiterpene moiety is still open to question, but E, E-famesyl cation has been suggested as one possibility and hedycarylol as a second. This moiety is dihydroagarofuran. Therefore, a for the sesquiterpene pyridine alkaloids is nicotinic acid and E, E-famesyl cation and, controversially, hedycaryol. The /3 is amacrocycling ring formation substance (two moieties), from which the alkaloid forms (Figure 62). 

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

The biochemical model contains the pathways of the enzymatic reactions in the synthetic routes. Model can be constructed for each alkaloid. Eigure 75 presents biochemistry model of Catharantus alkaloids. The most important enzymes on this model are TDC (tryptophan decarbocylase), GlOH (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... 

Chemical model of indole alkaloid formation in Catharanthus roseus. 124... 

Another theory of mental illness postulates endogenous alkaloid formation. Aldehydes formed by oxidation of catecholamines as well as formaldehyde and acetaldehyde are present in tissues in small amounts. Condensation with amines could generate Schiff bases and alkaloids as in Fig. 25-10. This "plant chemistry" is spontaneous and can apparently take place in the brain, where it may have a potent effect. 

The crude enzyme preparation was found to catalyse the conversion of cadaverine (16) mainly into 17-oxosparteine (27) in the presence of pyruvic acid. The pyruvic acid served as a receptor for the amino-groups of (16) in a transamination reaction, having manifestly a close relationship to alkaloid formation.11 Diamine oxidase activity might have been expected to account for the... 

The isolation of the cDNAs encoding the enzymes involved in diverse isoquinoline alkaloid formation in plants and microorganisms allowed the first metabolic engineering routes to be developed and paved the way for new ways of future production of isoquinoline alkaloids. For instance, transgenic opium poppy plants were created in which codeinone reductase was suppressed by RNAi, resulting in the substitution of morphine synthesis with the non-narcotic precursor reticuline [110]. In a similar approach, RNAi suppression or overexpression of salutaridinol 1-0-acetyltransferase in opium poppy led to accumulation of salutaridine or increase of morphine, codeine and thebaine content [111], suppression of the BBE led to accumulation of berberine in California poppy cells [112],... 

FRISCHKNECHT, P.M., BATTIG, M., BAUMANN, T.W., Effect of drought and wounding stress on indole alkaloid formation in Catharanthus roseus. Phytochemistry, 1987,26,707-710. 

Mammalian alkaloids, 119-172, see also Mammalian isoquinoline alkaloids formation as therapeutic concept, 168-169 Pictet-Spengler cyclization, 120-121, 123-124... 

Figure 1.7 Example of the complicated biochemistry and physiology of alkaloid formation quinolizidine alkaloids (QAs) in lupins (genus Lupinus, Fabaceae). QAs are formed in ieaf chioropiasts and exported via the phloem all over the plant. QAs predominantly accumulate in vacuoles of epidermal tissue. Organs important for survival and reproduction, such as flowers and seeds, store especially high amounts of defence alkaloids. (See Plate 6 in colour plate section.)...

Two enz)mies of pyrrolidine alkaloid formation responsible for the conversion of putrescine to the N-methylpyrrolinium ion have been investigated in some detail. PMT, partially purified from cultures of Hyoscyamus niger and fully characterized from Datura stramonium, has been cloned by differential screening of complementary deoxyribonucleic acid (cDNA) libraries from high- and low-nicotine-yielding N. tabacum plants (Hibi et ah, 1994). The enzyme shows considerable sequence homology to spermidine synthase but is distinct from this enz)mie as it only shows PMT activity when expressed in Escherichia coli. MPO has been isolated in pure form from N. tabacum transformed root cultures (McLauchlan et ah, 1993). It is quite widely spread in... 

Fig. 10. In vitro activities of the enzymes of alkaloid biosynthesis and rates of alkaloid formation in hyphae of Penicitlium cyclopium (75). Cultivation conditions were as described in the legend to Fig. 9. At the times indicated by arrows, cycloheximide (100 p.g/ml) was added to the culture medium. All values are in units per 1 cm of culture area. (A) Anthranilate adenylyltransferase (AA) (100 = 5.6 pkat) ( ) cyclopeptine dehydrogenase (CD) (100 = 40 pkat) ( ) dehydrocyclopeptine epoxidase (DE) (100 = 0.42 pkat) (A) cyclopenin m-hydroxylase (CH) (100 = 12 pkat) (O) cyclopenin-cyclopenol formation in vivo (100 = 9 pmol/sec).

Coordination of Alkaloid Formation with Biosynthesis of Precursor Amino Acids... 

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