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Due to the scarcity of C–F bond-forming enzymatic reactions in nature and the contrasting prevalence of organofluorine moieties in bioactive compounds, developing biocatalytic fluorination reactions represents a pre-eminent challenge in enzymology, biocatalysis and synthetic biology. Additionally, catalytic enantioselective C(sp3)–H fluorination remains a challenging problem facing synthetic chemists. Although many non-haem iron halogenases have been discovered to promote C(sp3)–H halogenation reactions, efforts to convert these iron halogenases to fluorinases have remained unsuccessful. Here we report the development of an enantioselective C(sp3)–H fluorination reaction, catalysed by a plant-derived non-haem enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), which is repurposed for radical rebound fluorination. Directed evolution afforded a C(sp3)–H fluorinating enzyme ACCOCHF displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the iron centre, underscoring the importance of substrate tunnel engineering in non-haem iron biocatalysis. Computational studies reveal that the radical rebound step with the Fe(III)–F intermediate has a low activation barrier of 3.4 kcal mol−1 and is kinetically facile. 

Abstract The effective design of combinatorial libraries to balance fitness and diversity facilitates the engineering of useful enzyme functions, particularly those that are poorly characterized or unknown in biology. We introduce MODIFY, a machine learning (ML) algorithm that learns from natural protein sequences to infer evolutionarily plausible mutations and predict enzyme fitness. MODIFY co-optimizes predicted fitness and sequence diversity of starting libraries, prioritizing high-fitness variants while ensuring broad sequence coverage. In silico evaluation shows that MODIFY outperforms state-of-the-art unsupervised methods in zero-shot fitness prediction and enables ML-guided directed evolution with enhanced efficiency. Using MODIFY, we engineer generalist biocatalysts derived from a thermostable cytochromecto achieve enantioselective C-B and C-Si bond formation via a new-to-nature carbene transfer mechanism, leading to biocatalysts six mutations away from previously developed enzymes while exhibiting superior or comparable activities. These results demonstrate MODIFY’s potential in solving challenging enzyme engineering problems beyond the reach of classic directed evolution. 

Abstract Cooperative catalysis with an enzyme and a small‐molecule photocatalyst has recently emerged as a potentially general activation mode to advance novel biocatalytic reactions with synthetic utility. Herein, we report cooperative photobiocatalysis involving an engineered nonheme Fe enzyme and a tailored photoredox catalyst to achieve enantioconvergent decarboxylative azidation, thiocyanation, and isocyanation of redox‐active esters via a radical mechanism. We repurposed and further evolved metapyrocatechase (MPC), a nonheme Fe extradiol dioxygenase not previously studied in new‐to‐nature biocatalysis, for the enantioselective C─N₃, C─SCN, and C─NCO bond formation via an enzymatic Fe─X intermediate (X═N₃, NCS, and NCO). A range of primary, secondary, and tertiary alkyl radical precursors were effectively converted by our engineered MPC, allowing the syntheses of organic azides, thiocyanates, and isocyanates with good to excellent enantiocontrol. Further derivatization of these products furnished valuable compounds including enantioenriched amines, triazoles, ureas, and SCF₃‐containing products. DFT and MD simulations shed light on the mechanism as well as the binding poses of the alkyl radical intermediate in the enzyme active site and the π‐facial selectivity in the enantiodetermining radical rebound. Overall, cooperative photometallobiocatalysis with nonheme Fe enzymes provides a means to develop challenging asymmetric radical transformations eluding small‐molecule catalysis.