De novo Design of a Vanadium Haloperoxidase

Organohalogens are an essential class of compounds for both synthetic chemistry and natural systems, yet the design of halogenases to catalyze carbon–halogen (C–X) bond formation has remained elusive. Vanadium-dependent haloperoxidases (V-HPOs) are attractive targets for such an effort, but they pose an unusual design problem: the vanadate (VO ₄ ³⁻ ) cofactor is an anionic oxometalate that must be held by a precisely arranged network of hydrogen-bonding and electrostatic interactions rather than by the strong coordinate-covalent bonds through which proteins bind cationic transition metals. We addressed this challenge by utilizing a machine-learning-guided workflow to design VAN1, a highly stable 20 kDa protein that binds VO ₄ ³⁻ and brominates activated aromatic substrates, including L -tyrosine. X-ray crystal structures of metal-bound VAN1 closely matched the computational model and confirmed the designed active site, and structure-guided mutagenesis improved VO ₄ ³⁻ affinity 15-fold and bromination activity 2.6-fold, in quantitative agreement with the resulting change in cofactor occupancy. VAN1 remains folded in neat organic solvents and is substantially more active in aqueous/organic mixtures than in water, representing an important advantage for synthetic applications. As the first example of a de novo enzyme that catalyzes C–X bond formation, VAN1 establishes a new starting point for V-HPO engineering and mechanistic studies.