CYTOCHROME P-450(TYR) IS A MULTIFUNCTIONAL HEME-THIOLATE ENZYME CATALYZING THE CONVERSION OF L-TYROSINE TO P-HYDROXYPHENYLACETALDEHYDE OXIME IN THE BIOSYNTHESIS OF THE CYANOGENIC GLUCOSIDE DHURRIN IN SORGHUM-BICOLOR (L) MOENCH

Cytochrome P-450(TYR), which catalyzes the N-hydroxylation of L-tyrosine in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench has recently been isolated (Sibbesen, O., Koch, B., Halkier, B. A., and Moller, B. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 92, 9740-9744). Reconstitution of the enzyme activity in lipid micelles containing cytochrome P-450, and NADPH-cytochrome P-450 oxidoreductase demonstrates that cytochrome P-450(TYR) catalyzes the conversion of L-tyrosine into p-hydroxyphenylacetaldehyde oxime. Earlier studies with microsomes have demonstrated that this conversion involves two N-hydroxylation reactions of which the first produces N-hydroxytyrosine. We propose that the product of the second N-hydroxylation reaction is N,N-dihydroxytyrosine. N,N-dihydroxytyrosine is dehydrated to 2-nitroso-3-(p-hydroxyphenyl) propionic acid which decarboxylates to p-hydroxyphenylacetaldehyde oxime. The dehydration and decarboxylation reactions may proceed non-enzymatically. The E/Z ratio of the p-hydroxyphenylacetaldehyde oxime produced by reconstituted cytochrome P-450(TYR) is 69:31. Lipid micelles made from L-alpha-dilauroyl phosphatidylcholine are more than twice as effective in reconstituting cytochrome P-450(TYR) activity as compared to other lipids. The K-m and turnover number of the enzyme is 0.14 mM and 200 min(-1), respectively, when assayed in the presence of 15 mM NaCl whereas the values are 0.21 mM and 230 min(-1) when assayed in the absence of added salt. The multifunctional nature cytochrome P-450(TYR) is confirmed by demonstrating that binding of L-tyrosine or N-hydroxytyrosine mutually excludes binding of the other substrate. These results explain why the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime as earlier reported (Moller, B. L., and Conn, E. E. (1980) J. Biol. Chem. 255, 3049-3056) shows the phenomenon of catalytic facilitation (''channeling''). Cytochrome P-450(TYR) is the first isolated multifunctional heme-thiolate enzyme from plants. N-Hydroxylases of the cytochrome P-450 type with high substrate specificity have not previously been reported.