Engineering the Reaction\nPathway of a Non-heme Iron\nOxygenase Using Ancestral Sequence Reconstruction

Non-heme iron (Fe<sup>II</sup>), α-ketoglutarate\n(α-KG)-dependent\noxygenases are a family of enzymes that catalyze an array of transformations\nthat cascade forward after the formation of radical intermediates.\nAchieving control over the reaction pathway is highly valuable and\na necessary step toward broadening the applications of these biocatalysts.\nNumerous approaches have been used to engineer the reaction pathway\nof Fe<sup>II</sup>/α-KG-dependent enzymes, including site-directed\nmutagenesis, DNA shuffling, and site-saturation mutagenesis, among\nothers. Herein, we showcase a novel ancestral sequence reconstruction\n(ASR)-guided strategy in which evolutionary information is used to\npinpoint the residues critical for controlling different reaction\npathways. Following this, a combinatorial site-directed mutagenesis\napproach was used to quickly evaluate the importance of each residue.\nThese results were validated using a DNA shuffling strategy and through\nquantum mechanical/molecular mechanical (QM/MM) simulations. Using\nthis approach, we identified a set of active site residues together\nwith a key hydrogen bond between the substrate and an active site\nresidue, which are crucial for dictating the dominant reaction pathway.\nUltimately, we successfully converted both extant and ancestral enzymes\nthat perform benzylic hydroxylation into variants that can catalyze\nan oxidative ring-expansion reaction, showcasing the potential of\nutilizing ASR to accelerate the reaction pathway engineering within\nenzyme families that share common structural and mechanistic features.