De novo design of artificial haloperoxidase enzymes

Project Summary/Abstract Understanding the relationships between three-dimensional protein structure, metal reactivity, and catalytic function promises routes to new drugs and treatments, green chemical processes, and bespoke catalysts. However, the complexity of natural metalloenzymes makes the design of wholly new enzymes one of the greatest challenges in chemical biology. Thus, the de novo design of functional metalloproteins, in which both the protein scaffold and metal-containing active site are designed “from scratch”, presents the most stringent test of our understanding of protein folding and protein-metal structure-function relationships. Using new machine-learning tools for protein design and structure prediction, this work will investigate metalloenzyme structure-function relationships though the de novo design of vanadium- and heme-dependent haloperoxidase enzymes. These enzymes oxidize halides (X–) to X+ using H2O2 to promote halogen incorporation into diverse organic substrates. Halogen atoms often greatly increase the bioactivity of organic compounds, making these reactions important both for human health (e.g., pharmaceuticals, hormones, antibiotics) and the environment (e.g., toxic and ozone-depleting aerosols such as bromoform). The designed proteins will be expressed and characterized using a diverse array of structural, biophysical, and spectroscopic methods. Haloperoxidase activity will be measured, and the effects of primary and secondary sphere mutations will be studied. Potential alternative reactions catalyzed by the enzymes will be assayed, and the binding and activities of similar metal-oxo species (e.g., MoO42–, WO42–) will be investigated. By designing haloperoxidases using only minimal starting motifs, this work will provide valuable insights into the requisite structural and chemical features enabling efficient haloperoxidation catalysis.