Biosynthesis of Pyrrolidine Type Nicotinoids Fig 310

Nicotine is synthesized mainly in the roots (>97%) as has been proved, e.g., for N. tabacum and N. rustica (Dawson 1941; Dawson and Solt 1959). Besides its accumulation in the roots it is transported to the shoots in the xylem stream (Baldwin 1989). There it is accumulated especially in young leaves, stems, and reproductive organs as a defence agent. One of its precursors, the N-methyl-A1-pyrrolinium cation is also synthesized in the roots. The formation of this cation has been explained already before (Fig. 3.2).

The second precursor, nicotinic acid, necessary for the pyridine moiety is formed in the pyridine nucleotide cycle which is also yielding the coenzymes nicotinamide-adenine-dinucleotide (NAD) and NADP, respectively, in all organisms. This cycle is fed by quinolinic acid (pyridine-2,3-dicarboxylic acid) which is formed by cyclization of l-aspartic acid and 3-phosphoglyceraldehyde as has been discovered during research work with tobacco (Leete 1983 and references therein). This is of general significance since it turned out to be a new biosynthetic pathway to the ubiquitous nicotinic acid which is the route of plants in contrast to the route of mammals (degradation of l-tryptophan). Quinolinic acid is introduced into the pyridine nucleotide cycle by decarboxylation at C-2 and by the simultaneous formation of an N-glycosidic linkage yielding nicotinic acid mononucleotide. This reaction is catalyzed by quinolinic acid phosphoribosyltransferase (QPT) whose activity turned out to be very high in Nicotiana roots in contrast to leaves (Mann and Byerrum 1974), more evidence for the location of the biosynthesis of nicotine in the roots. QPT is considered to be the main regulatory enzyme for nicotinic acid production in nicotine biosynthesis (Bush et al. 1999 and references therein). Nicotinic acid can be considered as the aglycone of this N-glycoside; the free acid may be formed by an immediate hydrolysis (catalyzed by nicotinic acid mono-nucleotide glycohydrolase) as well as after going through the whole cycle via NAD (Bush et al. 1993, 1999 and references therein). Nicotinic acid had been first

Nicotine Biosynthesis
Fig. 3.10 Biosynthesis of the major tobacco alkaloids nicotine, anabasine, and anatabine

obtained in the nineteenth century by oxidation of nicotine during attempts to elucidate the structure of the alkaloid. It had been recognized already at that time as a pyridine carboxylic acid by Laiblin (1877, 1879). Based on the fact that this acid is also a natural product in plants discovered as a constituent of rice bran (Suzuki et al. 1912), it was proposed as a likely precursor of nicotine in Nicotiana tabacum (Winterstein and Trier 1931). Thirty years later it could be confirmed that nicotinic acid can function as a precursor; isotopic labelled nicotinic acid supplied to root cultures of N. tabacum (Dawson et al. 1960) as well as to those of N. glauca (Solt et al. 1960) yielded substantial incorporation. In a cell-free system obtained from N. glutinosa the reaction between the N-methyl-A1-pyrrolinium cation and nicotinic acid forming nicotine could be proved (Friesen et al. 1992 and references therein). It is assumed that nicotinic acid is first decarboxylated (Leete 1983). An enzyme system which catalyzes the oxygen-dependent release of the carboxyl group from nicotinic acid was found in the roots of N. rustica (Chandler and Gholson 1972). The point of attachment of the pyrrolidine ring to the pyridine skeleton turned out to be at C-3 which had been the point of attachment of the car-boxyl group before (Scott and Lynn 1967). It was proposed that the nicotinic acid is activated by reduction to 3,6-dihydronicotinic acid (Leete and Mueller 1982; Leete 1983) which may be a genuine precursor to react with the N-methyl-A1-pyr-rolinium cation. Both last steps in nicotine biosynthesis, the condensation with this cation as well as the decarboxylation, are catalyzed by nicotine synthase (Friesen and Leete 1990). These reactions may proceed via 1,2-dihydropyridine [for comparison see biosynthesis of anabasine (Fig. 3.10)].

Nornicotine in N. glutinosa and in N. glauca is formed only in the leaves and at the expense of nicotine translocated from the roots as could be proved by reciprocal graft combinations with tomato (Solanum lycopersicum) shoots/roots (Dawson 1945). Already in this early report it was speculated that nicotine is converted to nor-nicotine "probably by transmethylation". However, according to a proposal of Leete (1977), N'-formylnornicotine may be formed by oxidation of the N-methyl group of nicotine followed by oxidation to nornicotine. Later a partial characterisation of nicotine N-demethylase from microsomes of N. otophora was documented. Demethylation was interpreted to be associated with cytochrome P-450 rather than achieved by transmethylation (Bush et al. 1999 and references therein). The enzyme turned out to be NADPH-dependent in cell-free preparations from cell cultures of N. tabacum (Hao and Yeoman 1996b). Recently, it has been proved that CYP82E4 is involved in the metabolic conversion of nicotine to nornicotine in tobacco (Siminszky et al. 2005).

Alternatively, it was proposed that N-formylnornicotine is formed by formylation of nornicotine (Burton et al. 1988). Such an acylation of nornicotine might be supported by the fact that similarities with the profile of the formation for N'-acetylnornicotine could be observed. Recently, this alternative mechanism was supported additionally by a study with cell suspension cultures of N. plumbaginifolia fed with [13C,2H3-methyl]nicotine or [1'-15N]nornicotine. It could be demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation: (i) nornicotine turned out to be derived directly from nicotine and (ii) it was evident that N'-formylnornicotine was one metabolite of nornicotine (Bartholomeusz et al. 2005). The authors concluded that the most probable mechanism is an oxidative elimination of the N'-methyl group of nicotine, i.e., via a putative N'-hydroxymethylnornicotine which could be spontaneously decomposed into nornicotine and formaldehyde. Possibly different mechanisms may occur in different Nicotiana species.

N'-Acylated nornicotines discovered in N. tabacum are distributed within the leaf matrix (Bush et al.1993). However, their more complex congeners discovered in the Repandae species are synthesized in the trichomes on the leaf surface as reported for N. stocktonii and "the novel alkaloid N-hydroxyacylnornicotine". The fatty acid composition was not elucidated for the first; apparently "the novel alkaloid" had been a mixture of congeners (Zador and Jones 1986) which could be separated and resolved soon (see Sect. 3.3.1). Its synthesis had direct connection with the pool of nicotine produced in the root, transported to the aerial parts, and converted in the leaf to nornicotine. The latter alkaloid appeared to be the substrate of an acyltransferase which apparently is not very specific with respect to the length of the chain. After acylation the derivatives were secreted immediately into the exudate by the glandular cells of the trichomes. The long chain fatty acids needed for the acylation are products of the chloroplasts (Stumpf and Jones 1963).

The minor pyrrolidine-type nicotinoids, i.e., cotinine, nicotyrine, myos-mine, N-methylmyosmine, and the nicotine-N'-oxides, are formed by simple chemical oxidation of nicotine with oxygen in solution. Therefore it was assumed that the presence of these alkaloids in the living plant is not due to a formation catalyzed by enzymes (Leete 1983). Primary carbon oxidation of nicotine in plants leads to (i) nicotine-1'-iminium ion (N-methylmyosmine), (ii) nicotine-5'-iminium ion which may be a precursor of cotinine [this is assumed for the mammalian metabolism (Gorrod 1993)], and (iii) nicotine-N-methylen-iminium ion which may be a precursor of nornicotine via N-hydroxymethylnor-nicotine [this is also assumed for the mammalian metabolism (Gorrod 1993)] (Bush et al. 1999). Though cotinine is derived from the oxidation of nicotine (Burton et al. 1988 and references therein), very little conversion of nicotine to cotinine was found when administered to the living N. glauca plant (Leete and Chedekel 1974). Myosmine was formed via nornicotine (Leete and Chedekel 1974; Bush et al. 1993; Bartholomeusz et al. 2005).

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