Alkaloid, biosynthesis

Kuzmanov, B. and Dutschewska, H. 1982. Evolutionary patterns and alkaloid biosynthesis in... 

BIOCHEMICAL AND MEDICINAL CHEMISTRY 5.2.9.1 Indole Alkaloid Biosynthesis... 

SEQUENCE-BASED APPROACHES TO ALKALOID BIOSYNTHESIS GENE IDENTIFICATION... 

This chapter is an historical perspective with selected examples from our own laboratory, first at the University of Munich, and now at the Leibniz Institute of Plant Biochemistry in Halle, that reflect the changes in how we define and exploit sequence-based approaches to alkaloid biosynthesis gene identification. ... 

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. 

In summary, the de novo isolation of the cDNAs encoding enzymes of alkaloid biosynthesis is still achieved by using a variety of classical techniques, such as protein purification followed by partial amino acid sequence determination, and by newer techniques such as proteomics coupled to functional heterologous expression. The current status of cloned cDNAs specifically related to isoquinoline alkaloid biosynthesis is schematically presented in Figure 10.8. New additions to this list will certainly be made in the future as a result of a combination of approaches both new and old. 

KUTCHAN, T.M., DITTRICH, H., Characterization and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants, J. Biol. Chem., 1995,270,24475-24481. 

PAULI, H.H., KUTCHAN, T.M., Molecular cloning and functional heterologous expression of two alleles encoding (5)-Y-methylcoclaurine 3 -hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent monooxygenase of benzylisoquinoline alkaloid biosynthesis, Plant J., 1998,13, 793-801. 

MORISHIGE, T TSUJITA, T YAMADA, Y SATO, F., Molecular characterization of the S-adenosyl-L-methionine 3 -hydroxy-A-methylcoclaurine 4 -0-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica, J. Biol. Chem., 2000, 275, 23398-23405. 

Sequence-Based Approaches to Alkaloid Biosynthesis Gene Identification... 163 Toni M. Kutchan... 

D-Arginine and D-ornithine metabolism D-Alanine metabolism Glutathione metabolism Alkaloid biosynthesis I Alkaloid biosynthesis II Metabolism of Complex Carbohydrates Starch and sucrose metabolism Biosynthesis and degradation of glycoprotein... 

Mannich and Mannich-like reactions are widely used for the chemical synthesis of heterocycles, and in alkaloid biosynthesis in plants. One such reaction important in nature is a biological equivalent of the Pictet-Spengler tetrahydroisoquinoline synthesis (see Section 11.10.4), and offers a slight twist, in that the enol nucleophile is actually a phenol. 

Of the four major classes of biochemicals (carbohydrates, proteins, nucleic acids and lipids), experiments have shown that the first three classes could have arisen through prebiotic chemistry. Although the biosynthesis of many natural products can be traced back to acetate (e.g. fatty acids, terpenes and polyketide biosynthesis) or amino acids (e.g. alkaloid biosynthesis), there are many whose biosynthetic origins are either obscure or result from a complex combination of pathways (Fig. 2). 

Alkaloid biosynthesis needs the substrate. Substrates are derivatives of the secondary metabolism building blocks the acetyl coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid and 1-deoxyxylulose 5-phosphate (Figure 21). The synthesis of alkaloids starts from the acetate, shikimate, mevalonate and deoxyxylulose pathways. The acetyl coenzyme A pathway (acetate pathway) is the source of some alkaloids and their precursors (e.g., piperidine alkaloids or anthraniUc acid as aromatized CoA ester (antraniloyl-CoA)). Shikimic acid is a product of the glycolytic and pentose phosphate pathways, a construction facilitated by parts of phosphoenolpyruvate and erythrose 4-phosphate (Figure 21). The shikimic acid pathway is the source of such alkaloids as quinazoline, quinoline and acridine. 

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