Cytochrome P-450 monooxygenases

Details Cytochrome P-450 monooxygenases Cytochrome P-450 monooxygenases

1996-08-08

1999-03-16

Marschel, Ardin H.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

435 71, 435 25, 536 221, C12Q 100, C12Q 126, G01N 3353, C07H 1900

Patent

active

058828516

DESCRIPTION:

BRIEF SUMMARY
This application is a .sctn. 371 of PCT/EP94/03938, filed on Nov. 28, 1994, and published on Jun. 15, 1995, as WO 95/16041, which claims priority of European Patent Application No. 93810860.2, filed on Dec. 8, 1993.
The present invention relates to genetic engineering in plants using recombinant DNA technology in general and to enzymes involved in the biosynthesis of cyanogenic glycosides and glucosinolates and genes encoding these enzymes in particular. The proteins and genes according to the invention can be used to improve the nutritive value or pest resistance of plants.
Cyanogenic glycosides constitute secondary plant metabolites in more than 2000 plant species. In some instances they are the source of HCN which can render a plant toxic if it is taken as food. For example the tubers of the cyanogenic crop cassava (Manihot esculenta) constitute an important staple food in tropical areas. However, the cyanogenic glycosides present in the tubers may cause cyanide poisoning in humans due to insufficiently processed cassava products. Other plant species whose enzymatic production of HCN accounts for their potential toxicity if taken in excess as food or used as animal feed include white clover (Trifolium repens), sorghum (Sorghum bicolor), linen flax (Linum usitatissimum), triglochinin (Triglochin maritima), lima beans (Phaseolus lunatus), almonds (Amygdalus) and seeds of apricot (Prunus), cherries and apple (Malus). The toxic properties could be reduced by blocking the biosynthesis of cyanogenic glycosides in these plants.
The primary precursors of the naturally occuring cyanogenic glycosides are restricted to the five hydrophobic protein amino acids valine, leucine, isoleucine, phenylalanine and tyrosine and to a single non-protein amino acid, cyclopentenylglycine. These amino acids are converted in a series of reactions to cyanohydrins which are ultimately linked to a sugar residue. Amygdalin for example constitutes the O-.beta.-gentiobioside and prunasin the O-.beta.-glucoside of (R)-mandelonitrile. Another example of cyanogenic glycosides having aromatic aglycones is the epimeric pair of the cyanogenic glycosides dhurrin and taxiphyllin which are to be found in the genus Sorghum and Taxus, respectively. p-Hydroxymandelo-nitrile for example is converted into dhurrin by a UDPG-glycosyltransferase. Similiar glycosyltransferases are believed to be present in most plants. Vicianin and lucumin are further examples for disaccharide derivatives similiar to amygdalin. Sambunigrin contains (S)-mandelonitrile as its aglycone and is therefore epimeric to prunasin.
Examples of cyanogenic glycosides having aliphatic aglycones are linamarin and lotaustralin found in clover, linen flax, cassava and beans. A detailed review on cyanogenic glycosides and their biosynthesis can be found in Conn, Naturwissenschaften 66:28-34, 1979, herein incorporated by reference.
The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been extensively studied (Halkier et al, `Cyanogenic glucosides: the biosynthetic pathway and the enzyme system involved` in: `Cyanide compounds in biology`, Wiley Chichester (Ciba Foundation Symposium 140), pages 49-66, 1988; Halkier and Moller, Plant Physiol. 90:1552-1559, 1989; Halkier et al, The J. of Biol. Chem. 264:19487-19494, 1989; Halkier and Moller, Plant Physiol. 96:10-17, 1990, Halkier and Moller, The J. of Biol. Chem. 265:21114-21121, 1990; Halkier et al, Proc. Natl. Acad. Sci. USA 88:487-491, 1991; Sibbesen et al, in: `Biochemistry and Biophysics of cytochrome P-450. Structure and Function, Biotechnological and Ecological Aspects`, Archakov, A. I. (ed.), 1991, Koch et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.053; and Sibbesen et al, 8th Int. Conf. on Cytochrome P450, Abstract PII.016). It has been found that L-Tyrosine is converted to p-hydroxy-mandelonitrile, the precursor of dhurrin with N-hydroxytyrosine and supposedly N,N-dihydroxytyrosine, 2-nitroso-3-(p-hydroxyphenyl)propionic acid, (E)- and (Z)-p-hydroxyphenylacetaldehyd oxime, and p-hydroxyph

REFERENCES:
Gabriac, et al., "Purification and Immunocharacterization of a Plant Cytochrome P450: The Cinnamic Acid 4-Hydroxylase", Archives of Biochemistry and Biophysics, 288(1): 302-309 (1991).
Halkier, et al., "The Biosynthesis of Cyanogenic Glucosides in Higher Plants", The Journal of Biological Chemistry, 264(33): 19487-19494 (1989).
Halkier, et al., "Biosynthesis of theCyanogenic Glucoside Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Partial Purification of the Enzyme System Involved", Plant Physiol., 90: 1552-1559 (1989).
Halkier, et al., "Cyanogenic glucosides: the biosynthetic pathway and the enzyme system involved", Department of Plant Physioology, Royal Veterinary and Agricultural University, 40: 49-66 (1988).
Halkier, et al., "The Biosynthesis of Cyanogenic Glucosides in Higher Plants", The Journal of Biological Chemistry, 265(34): 21114-21121 (1990).
Halkier, et al., "Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench", Plant Physiol., 96: 10-17 (1991).
Halkier, et al., "2-Nitro-3-(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane, two intermediates in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench", Proc. Natl. Acad. Sci., 88: 487-491 (1991).
Koch, et al., "The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantez).", Archives of Biochemistry and Biophysics, 292: 141-150 (1992).
Sibbesen et al., "Purification of the Hydroxylating Enzyme System Involved in the Biosynthesis of the Cyanogenic Glucoside Dhurrin in Sorghum Bicolor (l.) Moench", Biochemistry and Biophysics of Cytochrome P450, 232 (1991).

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