Transient metabolon dynamics tune enzyme 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. Whereas 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 toward productive cyclization, thereby suppressing derailment byproducts. 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 new avenues for synthetic biology and metabolic engineering.