Mapping Strigolactone Hydrolysis in DWARF14 via QM/MM String Method.

Strigolactone signaling is unusual among plant hormone pathways in that its receptor, DWARF14 (D14), also catalyzes the hydrolysis of the hormone. However, key aspects of this reaction remain unresolved including (i) whether hydrolysis proceeds via the canonical acyl substitution method initiated by nucleophilic attack on the butenolide (D-ring) or an alternative Michael addition at the enol-ether bridge, and (ii) the identity of the hydrolysis-induced covalent modification that promotes receptor activation. In this work we test these competing mechanistic hypotheses using QM/MM string method simulations of the D14-GR24 complex. Our simulations show that the acyl substitution pathway initiated by nucleophilic attack of the D-ring is strongly favored, supporting the canonical mode proposed by experimental studies. Furthermore, we find that multiple covalent adducts involving the butenolide ring and catalytic residues can form and interconvert along the reaction coordinate. These results suggest that the hydrolysis-induced covalent modification is not a single static species, but a dynamic ensemble of chemically related states. Our findings provide a mechanistic framework that reconciles previously conflicting experimental observations and clarifies the chemical basis by which strigolactone hydrolysis promotes receptor activation.