Abstract
The following compounds were tested as early intermediates in the conversion of tyrosine to p-hydroxymandelonitrile by a microsomal preparation from dark grown sorghum seedlings: p-hydroxyphenylacetamide, 1-nitro-2-p-hydroxyphenylethane, p-hydroxyphenyl-pyruvic acid oxime, tyramine, N-hydroxytyramine, and N-hydroxytyrosine. Of these, only N-hydroxytyrosine was metabolized to p-hydroxymandelonitrile. N-Hydroxytyrosine was produced from L-[U-14C]tyrosine in tracer experiments when unlabeled N-hydroxytyrosine was added as a trap. These data indicate N-hydroxytyrosine as the first intermediate in the biosynthesis of dhurrin, the cyanogenic glucoside of sorghum, and represent the first demonstration of the formation of an alpha-N-hydroxy-amino acid in a biological system. The enzyme system involved in this reaction was partially characterized with respect to substrate specificity and the effect of various inhibitors. The enzyme was shown to have properties different than those reported for the mammalian enzyme system(s) involved in the N-hydroxylation of amine drugs. The possible involvement of N-hydroxyamino acids in the biosynthesis of other secondary plant products is discussed.
Highlights
The following compounds were tested as early intermediates in the conversion of tyrosine to p-hydroxymandelonitrile by a microsomal preparation from dark grown sorghum seedlings: p-hydroxyphenylacetamide, 1-nitro-2-p-hydroxyphenylethane, p-hydroxyphenylpyruvic acid oxime, tyramine, N-hydroxytyramine, and
The production and utilization of N-hydroxytyrosine by the sorghum microsomal system strongly implies N-hydroxytyrosine as the first intermediate in the biosynthesis of the cyanogenic glucoside dhurrin
This interpretation is further supported by experiments with “02 which show the oxygen atom introduced into N-hydroxytyrosine to be derived from molecular oxygen [21]
Summary
Chemicals-L[LJ-14C]Tyrosine (specific activity 440 mCi/mmol) was purchased from New England Nuclear, Boston, MA. 1-Nitro-2-phydroxyphenylethane was synthesized from 1-nitro-2-hydroxyphenylethene [10]. p-Hydroxyphenylpyruvic acid oxime was synthesized by the method of Ahmad and Spenser (ll), except that the reaction time used was only 20 min. p-Hydroxyphenylacetaldehyde was synthesized by the method of Robbins [12], except that the synthesis was carried out under nitrogen to improve the yield and purity of product. The second and third techniques for measuring the metabolic activity of sorghum particles involved the use of “‘C-labeled substrates and the separation of intermediates and end products formed by TLC or gas-liquid chromatography (GLC), respectively These techniques were more informative in that they permitted the monitoring of individual steps of the pathway and the detection of intermediates when they accumulated in the reaction mixtures. This method effectively p-hydroxyphenylacetonitrile, separates p-hydroxyphenylacetaldoxime, and p-hydroxybenzaldehyde, the labeled products normally encountered in a sorghum particle mixture when [%]tyrosine is used as substrate This technique was not adequate in the present study since tyrosine, N-hydroxytyrosine, tyramine, and p-hydroxyphenylpyruvic acid oxime remain at the origin and p-hydroxybenzoic acid, which can be formed from p-hydroxybenzaldehyde, is only poarly separated from the origin. After lyophilization to dryness and silylation, the composition of the reaction mixtures was determined by the GLC/ GPC procedure
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