Biosynthesis nicotine

The V-methyl -A1 -pyrrol ini um cation is the last common intermediate in both TA and nicotine biosynthesis (Fig.7.4). V-Methy 1-A1 -pyrrolinium cation formation begins with the decarboxylation of ornithine and arginine by ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), respectively. Putrescine is formed... 

The first committed step in TA and nicotine biosynthesis is catalyzed by putrescine JV-methyltransferase (PMT) (Fig.7.4).82 A PMT cDNA isolated from tobacco showed extensive homology to spermidine synthase from mammalian and bacterial sources.83 A-Methylputrescine is oxidatively deaminated to 4-aminobutanal, which undergoes spontaneous cyclization to form the reactive A-methyl-A1-pyrrolinium cation. Although the enzymes involved are unknown, the A-methyl-A1-pyrrolinium cation is thought to condense either with acetoacetic acid to yield hygrine as a precursor to the tropane ring, or with nicotinic acid to form nicotine. 

ODC is not a rate-limiting step in nicotine biosynthesis.138 Overexpression of oat ADC in tobacco increased the accumulation of agmatine, but increased nicotine production was not detected.139... 

IMANISHI, S., HASHIZUME, K., NAKAKITA, M., KOJIMA, H MATSUBAYASHI, Y., HASHIMOTO, T., SAKAGAMI, Y., YAMADA, Y NAKAMURA, K., Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Mol. Biol., 1998,38,1101-1111. 

Nicotine biosynthesis is localized in the roots of Nicotiana plants, and the alkaloids are transported to the shoots in the xylem stream,70 mainly to young leaves and stems and the reproductive parts of the plant.72 At first glance, the costly transport mechanisms seem to be a disadvantage, as there is a time lag of 10 hr from time of induction until the increase of nicotine production.73 The roots, however, as the site of synthesis are well protected against herbivory and continue the production, even when up to 88% of the total leave area is removed.74 Optimization of the cost-value ratio seems to be the reason for the inducible defense acting as a cost-saving... 

Nicotiana alkaloids, which serve as chemical defence compounds, are synthesized in the roots and are transported to other plant organs, such as aerial parts, via the xylem. These alkaloids accumulate in vacuoles. PMT and A622 oxidoreductase are strongly expressed in the endodermis and outer cortex cells of tobacco root tips and to a lesser degree in other parts of the cortex and parenchyma cells surrounding the xylem (Shoji et ah, 2002). The localization of nicotine biosynthesis in the parenchyma cells surrounding the xylem may aid the loading of the xylem with nicotine. 

Nicotine biosynthesis also involves the incorporation of nicotinic acid (Fig. 2.2) (Robins et al., 1987), and the availability of this moiety can be as important in nicotine accumulation as that of the putrescine-derived portion. However, the enz)une responsible for the condensation of N-methylpyrrolinium with decarboxylated nicotinic acid, nicotine s)mthase (Friesen and Leete, 1990), was measured at only a very low level of activity, quite inadequate to account for the rates of nicotine accumulation observed in cultures. The molecular analysis of low-nicotine mutants of N. tabacum suggested the presence of regulatory genes (Me 1 and Me 2) governing the expression of nicotine bios)mthesis (Hibi et al, 1994). 

The overexpression of both trl and h6h from H. niger in N. tabacum plants have been reported [160]. Here, transgenic and control tobacco plants were fed with the tropane intermediate tropinone. Thus, tropine, the TRI-reaction product, was detected only in leaves of transgenic plants, with no correlation with trl transcript level and tropine amounts. Surprisingly, transgenic tobacco plants contained 3 to 13-fold more nicotine than wild type plants. Also, the presence of considerable amounts of nornicotine, myosmine, anabasine and anatabine contrasted with low levels in wild-type plants, indicating that the overexpression of trl and h6h perturb the normal nicotine biosynthesis when these new genes taken from a different metabolic pathway are introduced in tobacco [160]. 

In agreement with similar experiments on nicotine and hyoscyamine Datura metel) iV-methylputrescine (24) was a much better precursor for cuscohygrine and hyoscyamine in S. lurida than either putrescine or ornithine. [2- " C,(5- N]Omithine was incorporated into cuscohygrine with no loss of relative to and thus the nitrogen atom in cuscohygrine arises specifically from the 5-amino-group of ornithine. It was concluded from these data that decarboxylation and iV-methylation precede cyclisation to the N-methyl-pyrrolidine ring, with 5-Af-methylaminobutyraldehyde (25) or its cyclic form (26) as an intermediate a similar role for the latter compound has been demonstrated in nicotine biosynthesis. ... 

Additional studies investigating nicotine and its biological effects are numerous a few examples will be listed here. The damage induced increase in alkaloid production in Nicotiana, and its mechanism have been investigated [44,45], Nicotine biosynthesis continues to be studied [46,47] and has included the use of NMR [48]. A synthesis of 1 from 3,3 -dipyridyl was reported [49]. Nicotine showed potential as a molluscicide [50], it was a potent inhibitor of TAK-induced activation of polymorphonuclear leukocytes [51], and it caused reduction of herpes simplex virus type 1 production, as well as reduction of viral attachment to cell membranes [52]. Mechanisms involved in the behavioral and cognitive effects of nicotine have been investigated [53]. 

The stereochemistry of the last step in nicotine biosynthesis (Scheme 1) follows from the known stereochemistry of the alkaloid. It has been found that treating callus of Nicotiana tabacum... 

The manner in which the two nicotinic acid units may be joined (Scheme 1) is suggested by analogy with the probable intermediacy of a dihydronicotinic acid in nicotine biosynthesis. It is further suggested that anatalline (4) and nicotelline (5) are trimers of the dihydro-derivative (7). ... 

Kisaki, T.S., S. Mizusaki, and E. Tamaki Y-Methylaminobutyraldehyde, a new intermediate in nicotine biosynthesis Arch. Biochem. Biophys. 117 (1966) 677-678. 

Mizusaki, S., Y. Tanabe, M. Noguchi, and E. Tamaki Changes in the activities of ornithine decarboxylase, putrescine A-methyltransferase and A-methyl putrescine oxidase in tobacco roots in relation to nicotine biosynthesis Plant Cell Physiol. 14 (1973) 103-110. 

Partially in an endeavour to define the stereochemical requirements for enzyme activity in nicotine biosynthesis, C-methyl derivatives of (141), namely (144),... 

Nicotine.—Nicotine (33) is assembled in Nicotiana species from nicotinic acid (31) and N-methyl-A -pyrroline (32). Administration to Nicotiana plants of 5-fluoronicotinic acid and derivatives of (32) methylated at C-2 and C-3 has resulted in the formation in vivo of unnatural nicotine analogues.In contrast, 4-methylnicotinic acid has been found not to be transformed in vivo into 4-methylnicotine, presumably because this particular methyl group interferes sterically with the appropriate enzyme reactions involved in nicotine biosynthesis. ... 

PMT and H6H, which catalyze the first and last steps, respectively, in the biosynthesis of the tropane alkaloid scopolamine (Scheme 3), were localized to the pericycle in the roots of A. belladonna and Hyoscyamus muticus (Fig. 2A) 132,145). PMT also catalyzes the first step in nicotine biosynthesis (Scheme 3) and has been localized to the endodermis, outer cortex, and xylem in N. sylvestris (244,245). In contrast, TR-I, an intermediate enzyme in the tropane alkaloid pathway, resides in the endodermis and nearby cortical cells (Fig. 2A) (135) thus, intermediates of tropane alkaloid metabolism must also be transportai between cell types. The biosynthesis and storage of acridone alkaloids were also associated with endodermis in Ruta graveolens (246). 

The intact incorporations into nicotine 6.71) of A -methylputrescine 6.62) and A -methyl-A -pyrroline 6.64) further define the pathway to this alkaloid [significantly label from C-2 of the precursor 6.64) appeared at C-2 of the alkaloid]. N-Methyl-A -pyrroline 6.64) was isolated in radioactive form after feeding, for example, radioactive ornithine to Nicotiana species, thus establishing its role as an intermediate in nicotine biosynthesis. Additional persuasive evidence comes from the isolation from Nicotiana of an enzyme which catalyses the conversion of putrescine... 

In a rather obscure publication of the Hatano Tobacco Experiment Station, Yoshida (1973) determined the activities of enzymes catalyzing nicotine biosynthesis (see Chapter 6 for a further discussion of the biosynthesis) in the roots of tobacco plants. His experiments included plants that... 

Putrescine A-methyltransferase (PMT) catalyzes the first committed step in TPA and nicotine biosynthesis, the SAM-dependent... 

The niclnic2 double mutant has highly reduced nicotine contents (about 5% of wild type) and strongly decreased expression levels of nicotine biosynthesis genes. The genes corresponding to the nic loci have not been cloned yet. 

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