Project Summary Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen substitution. The importance of halogenation and limitations associated with current halogenation methods prompted us to develop new halogenation catalysts. This proposal outlines the evolution of flavin dependent halogenases (FDHs) and Fe(II) α-ketoglutarate dependent halogenases (FeDHs) for a range synthetic methods involving selective halogenation and related (i.e. pseudohalogen) atom transfer reactions. Work completed in the concluding project period included the first first examples of FDH directed evolution to improve the substrate scope, selectivity, and reactivity of these enzymes for practical synthetic applications. Structural, kinetic, and computational methods have since been used to provide rigorous characterization of the engineered enzymes and to inform subsequent engineering efforts. In the proposed project period, more advanced synthetic applications of these enzymes, including halogenation of electron deficient substrates, sequential halogenation/cross-coupling for lead diversification, and enantioselective halogenation will be pursued. In a major new direction, FeDHs will also be evolved to catalyze non-directed site-selective sp3 C-H halogenation, azidation, and other atom transfer reactions. These FDH and FeDH efforts will involve new advances in genome mining and directed evolution methodology to expedite the identification and optimization of enzymes for non-native catalysis, C-H bond functionalization, and synthetic applications.