2-NITRO-3-(PARA-HYDROXYPHENYL)PROPIONATE AND ACI-1-NITRO-2-(PARA-HYDROXYPHENYL)ETHANE, 2 INTERMEDIATES IN THE BIOSYNTHESIS OF THE CYANOGENIC GLUCOSIDE DHURRIN IN SORGHUM-BICOLOR (L) MOENCH

The biosynthetic pathway for the cyanogenic glucoside dhurrin derived from tyrosine has been studied in vitro by using [O-18]oxygen and a microsomal enzyme system obtained from etiolated sorghum seedlings. The products formed were purified by HPLC and TLC, and the incorporation of [O-18]oxygen was monitored by mass spectrometry. In the presence of NADPH and [O-18]dioxygen, L-tyrosine is converted to (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime with quantitative incorporation of an [O-18]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 [O-18]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 [O-18]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.