A novel cytochrome P450, CYP3201B1, is involved in (R)‐mandelonitrile biosynthesis in a cyanogenic millipede

Specialized arthropods and more than 2500 plant species biosynthesize hydroxynitriles and release hydrogen cyanide as a defensive mechanism. The millipede Chamberlinius hualienensis accumulates (R)‐mandelonitrile as a cyanide precursor. Although biosynthesis of hydroxynitriles in cyanogenic plants and in an insect are extensively studied, (R)‐mandelonitrile biosynthesis in cyanogenic millipedes has remained unclear. In this study, we identified the biosynthetic precursors of (R)‐mandelonitrile and an enzyme involved in (R)‐mandelonitrile biosynthesis. Using deuterium‐labelled compounds, we revealed that (E/Z)‐phenylacetaldoxime and phenylacetonitrile are the biosynthetic precursors of (R)‐mandelonitrile in the millipede as well as other cyanogenic organisms. To identify the enzymes involved in (R)‐mandelonitrile biosynthesis, 50 cDNAs encoding cytochrome P450s were cloned and coexpressed with yeast cytochrome P450 reductase in yeast, as cytochrome P450s are involved in the biosynthesis of hydroxynitriles in other cyanogenic organisms. Among the 50 cytochrome P450s from the millipede, CYP3201B1 produced (R)‐mandelonitrile from phenylacetonitrile but not from (E/Z)‐phenylacetaldoxime, whereas plant and insect cytochrome P450s catalysed the dehydration of aldoximes and hydroxylation of nitriles. CYP3201B1 is not phylogenetically related to cytochrome P450s from other cyanogenic organisms, indicating that hydroxynitrile biosynthetic cytochrome P450s have independently evolved in distant species. Our study will shed light on the evolution of cyanogenesis among plants, insects and millipedes. DATABASE: Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers LC125356–LC125405.