Cyanogenic glucosides (CNglcs) are β-glucosides of α-hydroxynitriles derived from different amino acids. They are well known and widespread defence compounds due to the release of toxic hydrogen cyanide upon degradation. They are widespread in plants, but are also present in some Arthropod species, mainly within butterflies and moths. Some moths and butterflies are able to biosynthesize the aliphatic CNglcs linamarin (from valine) and lotaustralin (from isoleucine) de novo, while others sequester them and other related CNglcs from their food plants. Within Zygaenoidea and Papilionoidea both strategies are working simultaneously for many species in the Zygaena and Heliconius genera.

The CNglc biosynthetic pathway has been characterised from many plant species, and has clearly evolved convergently in insects (Jensen et al. 2011). The biosynthetic pathway was elucidated in the burnet moth, Zygaena filipendulae, and consists of three enzymes: two P450 enzymes, CYP405A2 (CYP4 clan), CYP332A3 (CYP3 clan) and a glucosyl transferase, UGT33A1. Heliconius butterflies also produce linamarin and lotaustralin and have close homologs to CYP405A2 and CYP332A3.

The analysis of CYP405 and CYP332 genes in Lepidoptera (Zagrobelny et al., 2018) shows that they are members of relatively small subfamilies present in both cyanogenic and noncyanogenic species. In Spodoptera exigua, CYP332A1 metabolizes chlorpyrifos (Bo et al., 2020). The ability to make cyanogenic glucosides may have been derived from an ancestral ability to detoxify aldoximes (Zagrobelny et al., 2018). Later the CYP405s evolved to generate aldoximes, and a UGT was recruited into the pathway to establish de novo biosynthesis of CNglcs.

Yamaguchi and Asano (2025) studied FMOs and P450s from Chamberlinius hualienensis to identify the enzymes involved in cyanogenesis (see figure below adapted from that paper). An FMO catalyzes the first step, formation of aldoximes. This reaction is catalyzed by CYP405A2 in Zygaena.

Two P450s, CYP4GL4 (clan 4) and CYP30008A2 (clan 2) were shown to convert aldoximes to nitrile. This is homologous to the “first half” of the reaction catalyzed by CYP332A3 in Zygaena.

CYP3201B1 (clan 2) is a phenylacetonitrile hydroxylase which makes (R)-mandelonitrile (Yamaguchi et al., 2017, Yamaguchi and Asano, 2025)). This reaction is homologous to the “second half” of the multistep reaction catalyzed by CYP332A3 and CYP71E1, and is identical to the reaction of CYP71B103 during synthesis of prunasin in Eucalyptus (Hansen et al., 2018).