Cyanogenic glucosides in cassava and sorghum: the biosynthetic pathway and the enzyme system involved.

The biosynthesis of the cyanogenic glucosides linamarin/lotaustralin and dhurrin has been studied in vitro using microsomal enzyme systems obtained from etiolated seedlings of cassava (Manihot esculenta Crantz) and sorghum (Sorghum bicolor L.). A prerequisite to obtain active preparations from cassava seedlings is the complete removal of the endosperm pellicle covering the cotyledons before seedling homogenization. The enzyme system in cassava seedlings is located in the cotyledons and petioles. The linamarin and lotaustralin produced in these tissues is actively translocated to the hypocotyl and roots. In sorghum, the enzyme system is localized in the coleoptile and primary leaf and the dhurrin produced here is not translocated to other parts of the plant. The biosynthesis of linamarin, lotaustralin and dhurrin follows a general pathway involving the conversion of the respective parent amino acids valine, isoleucin and tyrosine to the corresponding N-hydroxyamino acid, alpha-nitro-carboxylic acid, aci-nitro compound, E-aldoxime, Z-aldoxime, nitrile and alpha-hydroxynitrile before final glucosylation to produce the cyanogenic glucoside. The pathway is highly channeled as demostrated by double-labelling experiments. The substrate specificity of the enzyme system is high at the level of the amino acid but less pronounced at the level of the aldoxime and the nitrile intermediates. The first N-hydroxylation step is the rate limiting reaction in the overall conversion. The biosynthetic pathway encompasses three hydroxilation reactions. The N-hydroxylation reaction producing the alpha-hydroxinitrile is dependent on cytochrome P-450 as demonstrated by photoreversible inhibition by carbon monoxide. The involvement of cytochrome P-450 is also demonstrated by inhibition of the in vitro biosynthetic reactions by mono-specific polyclonal antibodies raised towards NADPH-cytochrome P-450 oxireductase isolated from sorghum. The C-hydroxylation reaction converting P-450 hydroxy-phenyl-acetonitrile to p-hydroxymandelonitrile has been reconstitued in an in vitro system containing partially purified cytochrome P-450 from sorghum, NADPH-cytochrome.