Chemical protein synthesis enabled engineering of saccharide oxidative cleavage activity in artificial metalloenzymes

Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a facile one-pot semi-synthetic strategy for the construction of a type 2 copper center in the active site of azurin, which is achieved by substitution of Met121 with unnatural amino acid residues bearing a strong ligand N,N-bis(pyridylmethyl)amine (DPA) to mimic the function of typical histidine brace-bearing copper monooxygenases, such as lytic polysaccharide monooxygenases (LPMOs) involved in polysaccharide breakdown. The semi-synthetic proteins were routinely obtained in over 10-mg scales to allow for spectroscopic measurements (UV-Vis, CD, and EPR), which provides structural evidences for the Cu^II-DPA-modified azurins. 4-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a model substrate for the H₂O₂-driven oxidative cleavage reaction facilitated by semi-synthetic azurins, and the Cu^II-6 complex showed a highest activity (TTN 253). Interestingly, our semi-synthetic azurins were able to tolerate high H₂O₂ concentrations (up to 4000-fold of the enzyme), making them promising for practical applications. Collectively, we establish that chemical protein synthesis can be exploited as a reliable technology in affording large quantities of artificial metalloproteins to facilitate the transformation of challenging chemical reactions.