Cleavage of a carbon–fluorine bond by an engineered cysteine dioxygenase

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a posttranslationally generated Cys93–Tyr157 crosslinked cofactor. Here, we investigated this Cys-Tyr crosslinking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined crystal structures of both wild-type and engineered CDO variants in the purely uncrosslinked form and with a mature cofactor. Along with mass spectrometry and (19)F NMR, these data indicated that the enzyme could catalyze oxidative C–F or C–Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys-Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon–halogen bond activation.