Abstract
Flavonoids are essential for plant growth and environmental adaptation. Chalcone synthase (CHS) directs metabolic flux into flavonoid biosynthesis, but its catalytic promiscuity limits the biosynthetic efficiency. Although the chalcone isomerase-like protein (CHIL) has been characterized as physically interacting with and regulating CHS, the underlying mechanism remains elusive. Here, we report the crystal structure of the CHS-CHIL complex, revealing that CHIL modulates CHS function by gating the substrate-binding pocket entrance through its β-hairpin region. Molecular dynamics simulations indicate that regulation occurs early in the catalytic cycle, affecting substrate binding or early intermediate formation. CHIL enhances CHS activity and product specificity by promoting CoA release and stabilizing key amino acid residues. His36 within the β-hairpin is functionally critical; its substitution with leucine in Arabidopsis and other plant species markedly improves CHS catalytic efficiency and specificity. This regulatory mechanism is evolutionarily conserved across land plants, from bryophytes to angiosperms. Guided by evolutionary analysis, we engineer a CHIL variant (H36E/F37T) that significantly enhances CHS activity. Collectively, our findings establish a conserved binding-conformational regulation paradigm that governs metabolic flux into the flavonoid biosynthetic pathway and provides practical strategies for enhancing flavonoid production and composition in crops.