Transient dynamics of flavonoid metabolons tune chalcone synthase specificity

Cells achieve metabolic precision by assembling enzymes into dynamic complexes, but the regulatory mechanism of these metabolons is unclear. Here we characterize the chalcone synthase (CHS)–chalcone isomerase-like protein (CHIL) complex, a key component of flavonoid metabolons. While crystallography provides a static view, our analyses reveal that CHS undergoes rapid, reversible binding cycles with CHIL that regulate catalysis in real time. CHIL removes coenzyme A, an inhibitor of Claisen cyclization, transiently reshapes the CHS active site and guides the tetraketide intermediate towards productive cyclization, thereby suppressing derailment by-products. These findings demonstrate a previously unproven and generalizable regulatory effect in metabolons: guided active-site tuning via transient enzyme association. This concept enhances our understanding of metabolon function and opens avenues for synthetic biology and metabolic engineering. Enzyme assemblies can enhance metabolic efficiency, yet the ways in which transient interactions fine tune catalytic outcomes remain poorly understood. This study demonstrates that a flexible partner protein transiently remodels an enzyme’s active site to favour productive chemistry in a flavonoid metabolon.