Cytochrome P450 monooxygenases

Details Cytochrome P450 monooxygenases Cytochrome P450 monooxygenases

1999-11-12

2001-10-09

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C435S006120, C435S183000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023600, C536S024100, C800S301000

Reexamination Certificate

active

06300544

ABSTRACT:

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 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. 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-&bgr;-gentiobioside and prunasin the O-&bgr;-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-Hydroxymandelonitrile for example is converted into dhurrin (&bgr;-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) 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 P450. 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). L-Tyrosine is converted to p-hydroxy-mandelonitrile (the precursor of dhurrin), with N-hydroxytyrosine, N,N-dihydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyd oxime, and p-hydroxyphenylacetonitrile being intermediates. Two monooxygenases of the cytochrome P450 type are involved in this pathway. In cassava a similiar pathway involving cytochrome P450 dependent monooxygenases is used for the synthesis of linamarin and lotaustralin from valine and isoleucine, respectively (Koch et al, Archives of Biochemistry and Biophysics, 292:141-150, 1992). The complex pathway from L-tyrosine to p-hydroxy-mandelonitrile in
Sorghum bicolor
was demonstrated to require two multi-functional cytochrome P450 dependent monooxygenases only. The first enzyme, designated P450
TYR
, converts tyrosine to p-hydroxyphenylacetaldehyd oxime. The second enzyme, designated P450
OX
, converts the aldoxime to p-hydroxy-mandelonitrile. In view of the similiarities between the biosynthetic pathways of cyanogenic glucosides in different plants it is generally assumed that said pathways involve two multifuncitonal P450 dependent monooxygenases, P450
I
and P450
II
, which convert the precursor amino acid to the corresponding aldoxime and the aldoxime to the corresponding cyanohydrin, respectively. P450
I
is a specific enzyme which determines the substrate specificity and, thus, the type of glucoside produced, whereas P450
II
is expected to be less specific in converting a range of structurally different aldoximes into the corresponding cyanohydrin.
Glucosinolates are hydrophilic, non-volatile thioglycosides found within several orders of dicotyledoneous angiosperms (Cronquist, ‘The Evolution and Classification of Flowering Plants, New York Botanical Garden, Bronx, 1988). The occurance of cyanogenic glucosinolates and glucosides is mutually exclusive. The greatest economic significance of glucosinolates is their presence in all members of the Brassicaceae (order of Capparales), whose many cultivars have for centuries provided mankind with a source of condiments, relishes, salad crops and vegetables as well as fodders and forage crops. More recently, rape (especially
Brassica napus
and
Brassica campestris
) has emerged as a major oil seed of commerce. About 100 different glucosinolates are known possessing the same general structure but differing in the nature of the side chain. Glucosinolates are formed from protein amino acids either directly or after a single or multiple chain extension (Underhill et al, Biochem. Soc. Symp. 38:303-326, 1973). N-hydroxy amino acids and aldoximes which have been identified as intermediates in the biosynthesis of cyanogenic glycosides also serve as efficient precursors for the biosynthesis of glucosinolates (Kindl et al, Phytochemistry 7:745-756, 1968; Matsuo et al, Phytochemistry 11:697-701, 1972; Underhill, Eur. J. Biochem. 2:61-63, 1967). Cytochrome P450
I
involved in cyanogenic glycoside synthesis is thus functionally very similiar to the corresponding biosynthetic enzyme in glucosinolate synthesis, and is therefore expected to be a member of the same family of P450 enzymes. Thus we have isolated a cDNA clone from
Sinapis alba
encoding a P450 enzyme (SEQ ID NO:17) with 54% identity to P450
TYR
(CYP79) and catalyzing the first step in the biosynthesis of glucosinolates, that is the formation of the aldoxime from the parent amino acid. This cDNA clone shows approximately 90% identity to an Aribidopsis EST sequence (T42902) which strongly indicates that this cytochrome P450 enzyme is highly conserved in glucosinolate containing species.
The reduction of the complex biosynthetic pathway for cyanohydrins described above to the catalytic activity of only two enzymes, cytochrome P450
I
and P450
II
, allows for the manipulation of the biosynthetic pathway of cyanogenic glucosides in plants. By transfection of gene constructs coding for one or both of the monooxygenases a biosynthetic pathway for cyanogenic glucosides can either be modified, reconstituted, or newly established.
The modification or introduction of a biosynthetic pathway for cyanogenic glycosides in plants by methods known in the art is of great interest, since cyanogenic glycosides can be toxic to insects, acarids, and nematodes. Therefore, the modification, introduction or reconstitution of a biosynthetic pathway for cyanogenic glycosides in plants or certain plant tissues will allow to render plants unpalatable for insects, acarids or nemat

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