Enzymes of Quinolizidine Alkaloid Biosynthesis

The lysine decarboxylase from L. polyphyllus had a maximxim activity at pH 8.0, and was not inhibited by the ultimate alkaloids. 

17-oxosparteine and sparteine. The next step in the biosynthetic sequence is the transamination of cadaverine with pyruvate to yield alanine and 5-aminopentanal (40). It is proposed that this compound is enz3rme-bound (41), since none of the self-condensation products of this amino-aldehyde (the open-chain form of A -piperid-eine (39) ) such as tetrahydroanabasine or tripiperideine could be detected in the incubation mixture. The enzyme is called 17-oxosparteine synthase since no free intermediates between cadaverine and 17-oxosparteine could be isolated. The crude enzyme was first 

The multi-enzyme complex which affords the alkaloids has been isolated from the chloroplasts of L. polyphyllus leaves and L, albus seedlings . A diurnal fluctuation in the accumulation of the quinolizidine alkaloids was observed in L. polyphyllus and related species and could be related to the level of thioredoxin in the chloroplasts ° . 

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Wink, M. and Hartmann, T. 1980. Localization of enzymes of quinolizidine alkaloids biosynthesis in leaf chloroplast of Lupims polyphyllus Lindl. Plant Physiology, 70 lA-11. 

WINK, M., HARTMANN, T., Localization of the enzymes of quinolizidine alkaloid biosynthesis in leaf chloroplasts of Lupinus polyphyllus. Plant Physiol., 1982, 70, 74-77. 

Enzymes of Quinolizidine Alkaloid Biosynthesis.- In the past four years Hartmann, Wink, and their coworkers have obtained enzymes from Lupinus (especially L.polyphyllus) species which catalyse the formation of the tetracyclic quinolizidine alkaloids such as sparteine and lupanine from lysine. A novel biogenetic scheme has been proposed which accommodates these new results, and is consistent with previous biosynthetic studies on these alkaloids in intact plants . This new hypothesis (with some minor modifications by this reporter) is illustrated in Scheme 4. The first step in this sequence is the decarboxylation of lysine to yield cadaverine (38). This lysine decarboxylase was isolated from the chloroplasts of L. polyphyHub lea.ves, It is also present in... 

The biochemistry and molecular biology of quinolizidine alkaloid biosynthesis have not been fully characterized. Quinolizidine alkaloids are formed from lysine via lysine decarboxylase (LDC), where cadaverine is the first detectable intermediate (Scheme 6). Biosynthesis of the quinolizidine ring is thought to arise from the cyclization of cadaverine units via an enzyme-bound intermediate 176). LDC and the quinolizidine skeleton-forming enzyme have been detected in chlorop-lasts of L. polyphyllus 177). Once the quinolizidine skeleton has been formed, it is modified by dehydrogenation, hydroxylation, or esterification to generate the diverse array of alkaloid products. 

Quinolizidine Alkaloids.—Biosynthesis of quinolizidine alkaloids, e.g. sparteine (28), is from lysine (15) by way of cadaverine (16), as shown in Scheme 4. Three cadaverine units (as indicated by the thickened bonds) are required for the construction of alkaloids such as sparteine (28).10 Although something has been discerned about the biosynthetic relationships of various quinolizidine alkaloids, the nature of early intermediates beyond cadaverine has remained quite elusive.10 Exciting new results obtained with crude enzyme preparations from cell suspension cultures of Lupinus polyphyllus indicate why this is so. 

A -Piperideine (17) has been shown to be a precursor of quinolizidine alkaloids in whole plants (cf. Vol. 8, p. 3). However, neither it nor its self-condensation products could be detected as products in the enzymic reaction. [This conclusion is not completely unambiguous, albeit reasonably safe, because the products of the reaction of diamine oxidase, the first of which is (17), were simply compared with those of the alkaloid synthase reaction by g.l.c., and the products of the two reactions were found to be different].11 It seems likely at this stage that (17) is not normally implicated in quinolizidine biosynthesis but can be substituted for an enzyme-generated intermediate via its open form (32) (see Scheme 5). Since no intermediates earlier than (27) could be detected, it is suggested that biosynthesis in vitro and in vivo proceeds by a series of enzyme-linked intermediates (see Scheme 5), none of which is desorbed from the enzyme or enzyme-complex until (27) is liberated. However, in some plants, biosynthesis must stop with the liberation of a compound (31), having the lupinine skeleton... 

Quinolizidine Alkaloids.—Important new information (cf. Vol. 11, p. 4) has been obtained on the biosynthesis of quinolizidine alkaloids such as lupanine (27) in experiments with enzyme preparations from Lupinus polyphyllus cell suspension cultures26 and with chloroplasts.27 These alkaloids are formed from three molecules of lysine by way of cadaverine (25),1,2 and the enzymic evidence26,27 is that conversion of cadaverine into these alkaloids occurs without release of intermediates until 17-oxosparteine (26) is generated the enzyme is a transaminase and not a diamine oxidase. 

The enzyme, i.e. lysine decarboxylase, that is required for the conversion of lysine into cadaverine, and thus the first step of alkaloid biosynthesis, has been isolated from chloroplasts of L. polyphyllus,28 Like the majority of amino-acid decarboxylases, this enzyme is dependent on pyridoxal 5 phosphate. Its activity was found not to be affected by the presence or absence of quinolizidine alkaloids. Control of the enzyme by simple product feedback inhibition therefore seems unlikely. The operational parameters of this enzyme resemble those of the 17-oxosparteine synthase. Co-operation between the two enzymes would explain why cadaverine is almost undetectable in vivo. 

Unique subcellular compartmentation is also present in quinolizidine alkaloid biosynthesis, which occurs in the mesophyll chloroplasts of some legumes.158 One of the enzymes catalyzing the last two acylations of the pathway in Lupinus albus occurs in the cytoplasm, whereas the other resides in the mitochondria/59 Although the quinolizidine nucleus appears to be synthesized in the chloroplast, subsequent modifications can occur only after alkaloid intermediates are transported to the cytosol and mitochondia. Quinolizidine alkaloids appear to accumulate in vacuoles of epidermal cells where their defensive properties are most effective. 

Figure 4.16 Biosynthesis of quinolizidine alkaloids. Enzyme abbreviations ECT, p-coumaroyl-CoA (+)-epilupinine O-p-coumaryltransferase HMT/HLT, Tigloyl-CoA (-)-13a-hydroxymultiflorine/(+)-13a-hydroxylupanine-O-tigloyltransferase LDC, lysine decarboxylase. 

Scheme 4 Biosynthesis of the Quinolizidine Alkaloids via Enzyme-Bound Intermediates...

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