Structure-guided engineering of a plant dioxygenase for selective whole-cell optical discrimination of L-DOPA and dopamine

Selective discrimination between structurally related catecholamine metabolites remains a persistent challenge in biosensing. Here, we report the structure-guided engineering of Mirabilis jalapa DOPA 4,5-dioxygenase (MjDOD) for selective whole-cell optical discrimination of L-DOPA and dopamine. The 2.28 Å crystal structure of MjDOD revealed a non-heme iron-containing active site coordinated by three histidine residues together with second-shell residues that shape catecholic substrate recognition. Guided by structural analysis, ligand modeling, and mutational interrogation, we identified active-site determinants that govern discrimination between L-DOPA and dopamine. Among the engineered variants, N249D markedly suppressed reactivity toward L-DOPA while preserving productive turnover with dopamine, whereas complementary substitutions favored the native substrate. When deployed in Escherichia coli, these variants generated distinct absorbance-based whole-cell optical responses from closely related catecholic substrates. This work establishes an engineered plant dioxygenase scaffold as a selective recognition element for whole-cell biosensing and provides a structural framework for tuning metabolite discrimination in enzyme-based sensing systems.