Elucidating enzyme-substrate specificity through co-folding foundation model

Enzymatic catalysis relies on precise structural and chemical complementarity, yet systematically mapping enzyme-substrate interactions remains a critical bottleneck. While structure-aware methods have advanced functional annotation, their reliance on predefined binding pockets and rigid-body docking fails to capture the ligand-induced conformational changes essential for catalytic turnover. Here we introduce Boltz2ESI, an end-to-end framework that predicts enzyme-substrate interactions by leveraging structural knowledge learned by a biomolecular foundation model. Through native co-folding, the framework inherently captures active-site plasticity without requiring predefined pocket annotations. Integrating these learned biophysical priors with global evolutionary context and geometric molecular descriptors, Boltz2ESI consistently outperforms state-of-the-art sequence-based and rigid-docking approaches. Extensive validation demonstrates that the framework accurately discriminates tight sub-family specificities, enabling effective candidate prioritization for biosynthetic pathway elucidation, as demonstrated on the withanolide pathway. Ultimately, this structure-dynamic approach establishes an actionable foundation for accelerating rational biocatalyst discovery and large-scale pathway de-orphaning.