Alkaloid enzymology

Most of the recent progress in alkaloid enzymology has been made possible by purified enzymes that catalyze the respective steps of alkaloid biosynthesis. This is illustrated by the application of these techniques to alkaloid biosynthesis. For example, formation of ajmalicine (1) from its precursors, tryptamine (2) and secologanin (3), involves four key intermediates (Fig. 1.1). 

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. 

Waller, G. R. and Dermer, O. C. 1981. Enzymology of alkaloid metabolism in plants. In The Biochemistry of Plants. A Comprehensive treatise. Vol. 7. Secondary Plant Products (Conn, E. E., ed.), pp. 317 02. London Academic Press. 

Roberts, M. F. 1998. Production of alkaloids in plant cell culture. In Alkaloids. Biochemistry, Ecology, and Medicinal Applications (Roberts, M. F. and Wink, M., eds), pp. 159-197 and, Roberts, M. F. 1998. Enzymology of alkaloid biosynthesis. In Alkaloids. Biochemistry, Ecology, and Medicinal Applications (Roberts, M. F. and Wink, M., eds), pp. 109-146. New York - London Plenum Press. 

Kutchan, T.M., Dittrich, H., Bracher, D. and Zenk, M.H. (1991) Enzymology and molecular biology of alkaloid biosynthesis. Tetrahedron, 47,5945-54. 

De Luca, V. (1993) Enzymology of indole alkaloid biosynthesis, in Methods in Plant Biochemistry (ed. P.J. Lea), Vol. 9. Academic Press, London, pp. 345-67. 

The biosynthesis of monoterpenoid indole alkaloids has been intensively investigated since the sixties [76], The great interest in this group of alkaloids is due mainly to the cytotoxic vinblastine and vincristine, which are very useful anticancer drugs, but which are produced by the C. roseus plant in extremely low quantities. It is therefore not surprising that most of the biosynthetic and enzymology studies of monoterpenoid indole bases have been performed to elucidate the biosynthetic pathway of vinblastine and vincristine and its regulation in C. roseus plants. 

It is now apparent, however, that the Specialist Periodical Reports on "The Alkaloids", "Biosynthesis", "Terpenoids and Steroids" and "Aliphatic and Related Natural Product Chemistry" are not financially viable in their present book format and in 1984 they are to be replaced by a review journal to be called "Natural Product Reports". The new journal is to be published every two months and will continue to give comprehensive annual surveys of the four areas of natural product research additional articles on subjects not covered by existing Specialist Periodical Reports on topics such as chemotaxonom.y and enzymology and on advances in physical techniques will be included periodically and author and subject indices are to be provided. The flexible production timetable should ensure more rapid publication of manuscripts compared with S.P.R. s. In order to maintain continuity the S.P.R. Senior Reporters are members of the Editorial Board of Natural Products Reports and in the case of the alkaloid chapters it is expected that the present authors will contribute to the Journal. We all hope that the support we have received over the years from alkaloid chemists will be extended to this new and exciting venture. As always comments and suggestions will be much appreciated. 

Interest in enzyme stereospecificity and the stereochemistry of prochiral centres, such as the methylene groups of mevalonic acid, has necessitated more precise definitions of the stereochemistry of the various molecules involved and of the enzymological consequences. The use of multiply labelled mevalonic acid in terpenoid and steroid biosynthesis has been reviewed by Hanson. The Proceedings of the 1970 Phytochemical Society symposium have been published. They include a general discussion of terpenoid pathways of biosynthesis by Clayton and specific chapters on monoterpenoids, diterpenoids, eedysones, carotenoids, isoprenoid quinones, and chromanols. Other reviews concerning biosynthesis have appeared on furanocoumarins, indole alkaloids, monoterpenoids, and diterpenoids. ... 

De Luca V. Enzymology of indole alkaloid biosynthesis. In Lea PJ, editOT. Enzymes of Secondary Metabolism. Dey PM, Harbome JB, editos. Methods in Plant Biochemistry, voL 9. London, San Diego J few York, Boston, Sydney, Tokyo, Toronto Academic Press 1993. p. 345 8. 

Roberts MF. Enzymology of alkaloid biosynthesis. In Roberts MF, Wink M, editors. Alkaloids. Biochemistry, Ecology, and Medicinal Applications. New York, London Plenum Press 1998. p. 109-46. 

M.H. Zenk, Enzymology of benzylisoquinoline formation, in J.D. Phillipson, M.F. Roberts and M.H. Zenk (eds.), The Chemistry and Biology of Isoquinoline Alkaloids, Springer, Berlin, Heidelberg, New York, Tokyo (1985), pp. 240-256. 

As another example of the value of enzymological studies, the major enzymes of the benzylisoquinoline alkaloid pathway have also been isolated from plant tissue culture and studied by Zenk and co-workers (see Chapter 34) (Fig. 1.2). An enzyme, (S)-norlaudanosoline synthase, which catalyzes the conversion of dopamine and (3,4-dihydroxyphen-yl)acetaldehyde (9) into (S)-norlaudanosoline (10) has been purified about 40-fold from cell suspension cultures of Eschscholtzia tenuifolia (see also cluster 32 for up-dated in-... 

Wink, M. and T. Hartmann, Enzymology of quinolizidine alkaloid biosynthesis, in Natural Products Chemistry 1984 (14th lUPAC Symposium on the Chemistry of Natural Products) (R, I. Zalewski and J. J. Skolik, eds.), Elsevier, Amsterdam, 1984. 

Apart from the sizable primary literature on the biosynthesis of the tropanes, a substantial number of reviews on this topic has been published. The two most recent of these describe the work from the laboratory of the late Eddie Leete [6] and the encouraging results that have been obtained on an enzymological level [7]. Experiments aimed at elucidating the biosynthesis of the tropic acid moiety present in many tropane alkaloids have been reviewed recently by workers in the field [8] and will not be covered here. 

The experimental data accumulated so far for the origin of the acetate derived C3 chain of (1) are in agreement with this proposal. The presence of two bond-labeling patterns in (1) after administration of [1,2- C2]acetate has been interpreted hitherto as evidence for the presence of a racemic intermediate. This necessitated the postulation of a nonstereoselective enzymatic oxidation of (22) to (25) as discussed in detail above. However, this postulate in the face of the complete lack of enzymological data in its support, is not entirely satisfying. In contrast, the postulation of acetonedicarboxylic acid, or a derivative thereof, as a pathway intermediate does explain simply the labeling pattern in the C3 side-chain of (1) and a precedent has now been found in the biosynthesis of (27). Moreover, the hypothesis also explains why (21) is not incorporated into tropane alkaloids. 

Wink M, Hartmatm T (1985) Enzymology of quinolizidine alkaloid biosynthesis. In Zalewski RI, Skolik JJ (eds) Natural products cherrristry. Elsevier, Amsterdam, pp 511-520... 

Although the alkaloid field is a very old one, it is still in its infancy with regard to understanding its biosynthesis pathways enzymology and their regulation. Exploitation of the biotechnological potential of alkaloid biosynthesis has only just begun. 

Shough HR, Taylor EH (1969) Enzymology of ergot alkaloid biosynthesis. Part IV. Additional studies on the oxidation of agroclavine by horseradish peroxidase. Uoydia/J Nat Prod 32 315-326... 

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