Characterizing Y224 conformational flexibility in FtmOx1-catalysis using 19F NMR spectroscopy†

α-Ketoglutarate-dependent non-haem iron (αKG-NHFe) enzymes play a crucial role in natural product biosynthesis, and in some cases exhibiting multifunctional catalysis capability. This study focuses on αKG-NHFe enzyme FtmOx1, which catalyzes endoperoxidation, dealkylation, and alcohol oxidation reactions in verruculogen biosynthesis. We explore the hypothesis that the conformational dynamics of the active site Y224 confer the multifunctional activities of FtmOx1-catalysis. Utilizing Y224-to-3,5-difluorotyrosine-substituted FtmOx1, produced via the amber codon suppression method, we conducted ¹⁹F NMR characterization to investigate FtmOx1's structural flexibility. Subsequent biochemical and X-ray crystallographic analyses provided insights into how specific conformations of FtmOx1–substrate complexes influence their catalytic activities. These findings underscore the utility of ¹⁹F NMR as a powerful tool for elucidating the complex mechanisms of multifunctional enzymes, offering potential avenues for developing biocatalytic processes to produce novel therapeutic agents harnessing their unique catalytic properties.