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

Abstract

The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been studied in vitro by using [18O]oxygen and a microsomal enzyme system obtained from etiolated sorghum seedlings. The products formed were purified by HPLC and TLC, and the incorporation of [18O]oxygen was monitored by mass spectrometry. In the presence of NADPH and [18O]dioxygen, L-tyrosine is converted to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime with quantitative incorporation of an [18O]oxygen atom into the oxime function. The first step in this conversion is the N-hydroxylation of L-tyrosine to N-hydroxytyrosine. Administration of N-hydroxytyrosine as a substrate results in the production of 1-nitro-2-(p-hydroxyphenyl)ethane in addition to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, with quantitative incorporation of a single [18O]oxygen atom in all three products. These data demonstrate that the conversion of N-hydroxytyrosine to p-hydroxyphenylacetaldehyde oxime involves additional N-hydroxylation and N-oxidation reactions giving rise to the formation of 2-nitro-3-(p-hydroxyphenyl)propionate, which by decarboxylation produces aci-1-nitro-2-(p-hydroxyphenyl)ethane. Both compounds are additional intermediates in the pathway. The two [18O]oxygen atoms introduced by the N-hydroxylations are enzymatically distinguishable as demonstrated by the specific loss of the oxygen atom introduced by the first N-hydroxylation reaction in the subsequent conversion of aci-1-nitro-2-(p-hydroxyphenyl)ethane to (E)-p-hydroxyphenylacetaldehyde oxime. A high flux of intermediates through the microsomal enzyme system is obtained with N-hydroxytyrosine as a substrate. This renders the conversion of the aci-nitro compound rate limiting and results in its release from the active site of the enzyme system and accumulation of the tautomeric nitro compound.