The Role of Conformational Changes in TcmN Aromatase/Cyclase in Polyketide Biosynthesis

ABSTRACT Polyketide biosynthesis relies on the conformational adaptability of type II polyketide synthases and accessory enzymes, which direct chain folding and regiospecific cyclization. The aromatase/cyclase TcmN from Streptomyces glaucescensis catalyzes the first two ring closures of tetracenomycin C. Still, the molecular basis by which conformational dynamics regulate substrate binding and product release remains unresolved. Understanding how conformational transitions control ligand recognition and prevent aggregation is crucial for deciphering the molecular bases of polyketide biosynthesis and for guiding engineering strategies to synthesize novel natural products. Here, we investigated how ligand interactions modulate the conformational equilibrium of TcmN and the mechanistic consequences for catalysis. Using NMR spectroscopy (STD, CSP, relaxation dispersion), calorimetry, molecular docking, and microsecond-scale molecular dynamics simulations, we mapped the conformational ensembles of apo TcmN and its complexes with naringenin (a substrate/product analogue) and intermediate 12 (INT12). Apo TcmN samples both open and closed conformations. Naringenin preferentially stabilizes the closed state, a conformation thought to protect hydrophobic residues from solvent exposure. In contrast, INT12 shifts the equilibrium toward the open state, characterized by an expanded cavity that permits substrate entry, product release, and accommodation of extended intermediates. Hydrogen-bond analysis highlighted conserved catalytic residues (R82, E34, Q110, T133) as key anchors for productive poses. These results establish that TcmN functions through a ligand-gated breathing mechanism, in which successive intermediates selectively tune the cavity volume and shape, balancing catalytic efficiency with protection against aggregation. Conformational adaptability emerges as a central determinant of aromatase/cyclase function, providing molecular insights relevant for polyketide biosynthetic engineering. Graphical Abstract