Nanopore-enabled time-resolved monitoring of catecholamine-related phenylalanine metabolism

Catecholamines, including dopamine, noradrenaline (also known as norepinephrine) and adrenaline (also known as epinephrine), are essential regulators of neural and endocrine function. Although they are synthesized through a branched phenylalanine metabolic pathway, direct and dynamic monitoring of the enzymatic processes governing their production has remained challenging. Here we reconstitute the complete catecholamine biosynthetic pathway from phenylalanine to adrenaline in vitro using purified enzymes and cofactors, and couple this system with nanopore-based single-molecule sensing. By integrating two orthogonal molecular recognition modalities within a single nanopore platform, we achieve highly specific detection of all metabolic intermediates and end products throughout the pathway. Real-time monitoring enables direct observation of pathway progression and regulatory perturbations at single-molecule resolution. Using this approach, we show that 6-methylisothiocyanate disrupts catecholamine biosynthesis, providing mechanistic insight into how iodotyrosine dehalogenase 1 (DEHAL1) deficiency may compromise catecholamine production. Together, this integrated enzymology and label-free nanopore sensing platform enables time-resolved dissection of neurotransmitter metabolism and regulation, and offers a general framework for studying complex biochemical pathways with single-molecule resolution.