Cyanogenic glucosides are a class of specialized metabolites found in numerous crop plants with dhurrin being present in Sorghum bicolor. Dhurrin is formed from tyrosine in a pathway catalyzed by CYP79A1, CYP71E1, UGT85B1 and P450 oxidoreductase (POR). The three membrane bound proteins are situated in the ER membrane and the biosynthetic enzymes are thought to organize in enzyme complexes facilitating channeling of toxic and labile intermediates.In vivostudies based on transient expression in Nicotiana benthamiana demonstrated that all enzymes were active when expressed as eGFP fusion proteins and catalyzed efficient channeling of intermediates. Protein-protein interactions and
channeling was studiedin plantausing fluorescence lifetime imaging microscopy, fluorescence correlation spectroscopy and metabolite analysis. We demonstrate that the enzymes catalyzing dhurrin biosynthesis are organized within dynamic metabolons enabling plants to adapt to environmental challenges. Using the styrene maleic acid (SMA) copolymer, discrete lipid particles (SMALPs) were
excised from the ER membrane enabling purification of the dhurrin metabolon by affinity chromatography and characterization by mass spectrometry based proteomics. Functional importance of identified protein-protein interactions and lipid environment was studied by reconstitution of the dhurrin pathway in proteoliposomes. UGT85B1 binding to liposomes was dependent of the presence of CYP79A1 and CYP71E1. A model for the organization of the dhurrin metabolon in multi-enzyme clusters is presented. This SMALP nanodisc approach may be generally employed for detergent free isolation of entire
biosynthetic pathways organized within metabolons to identify enzymes catalyzing missing steps and to identify the presence of lipids stabilizing protein entanglement.

Talk on the P4 session: The plant endoplasmic reticulum: A dynamic multitasking organelle