4-hydroxyphenylacetaldoxime and dhurrin

Cytochrome P-450TYR, 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 Møller, B. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9740-9744). Reconstitution of the enzyme activity in lipid micelles containing cytochrome P-450TYR and NADPH-cytochrome P-450 oxidoreductase demonstrates that cytochrome P-450TYR 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-450TYR is 69:31. Lipid micelles made from L-alpha-dilauroyl phosphatidylcholine are more than twice as effective in reconstituting cytochrome P-450TYR activity as compared to other lipids. The Km 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-450TYR 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 (Møller, B. L., and Conn, E. E. (1980) J. Biol. Chem. 255, 3049-3056) shows the phenomenon of catalytic facilitation ("channeling"). Cytochrome P-450TYR 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.

Topics: Catalysis; Cytochrome P-450 Enzyme System; Edible Grain; Mixed Function Oxygenases; Nitriles; Oximes; Tyrosine

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (Møller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled.

Topics: Carbon Radioisotopes; Chromatography, High Pressure Liquid; Deuterium; Glucosides; Glycosides; Isomerism; Mass Spectrometry; Microsomes; Nitriles; Oximes; Plants; Radioisotope Dilution Technique; Tyrosine

The biosynthetic pathway for the cyanogenic glucoside, dhurrin, involves the following intermediates: L-tyrosine, N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile. N-Hydroxytyrosine and p-hydroxy-phenylacetonitrile produced from L-tyrosine by microsomes from seedlings of Sorghum bicolor are utilized more effectively as substrates than exogenously added N-hydroxytyrosine and p-hydroxyphenylacetonitrile. The minimum values for the channeling ratios are 25 for N-hydroxytyrosine and 115 for p-hydroxyphenylacetonitrile. On the other hand, p-hydroxyphenylacetaldoxime produced internally exchanges readily with exogenously added p-hydroxyphenylacetaldoxime. These results indicate that the biosynthetic pathway is catalyzed by two mutienzyme complexes or by two multifunctional proteins and explain why the rate of the overall sequential reaction starting from L-tyrosine is greater than the rates of reaction initiated later in the sequence with the known intermediates N-hydroxytyrosine and p-hydroxyphenylacetonitrile. Attempts to cross-link chemically the last enzyme in the pathway, a soluble UDP-glucose glucosyl-transferase, to the microsomal system were unsuccesful.

Topics: Carbon Radioisotopes; Glucosides; Glycosides; Isotope Labeling; Kinetics; Microsomes; Nitriles; Oximes; Plants; Tritium; Tyrosine