De novo Design of a Vanadium Haloperoxidase

Abstract

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 (VO43-) 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 VO43- 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 VO43- 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.