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

Abstract

Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93-Tyr157 cross-linked cofactor. Here, we investigated this Cys-Tyr cross-linking 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 the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 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.

Unleashing the Synthetic Power of Plant Oxygenases: From Mechanism to Application

“…Briefly, CDOs utilize a cupin fold with a three-His iron motif to which the L-Cys substrate coordinates via its thiol and amino groups (McCoy et al, 2006;Simmons et al, 2006). Mammalian CDOs also contain a conserved Cys(S)-Tyr(meta-C) cross-link in the active site (McCoy et al, 2006;Li et al, 2018b), believed to help stabilize reactive Fe-oxo species (Ye et al, 2007). PCOs, first identified in Arabidopsis, are linked to the regulation of hypoxic response and were recently shown to specifically oxidize the N termini of class VII Ethylene Response Factors (ERF-VIIs; Fig.…”

Enzymatic defluorination of fluorinated compounds

“…Enzyme engineering is a powerful tool for providing new functions to the biological catalysts [38][39][40]. In a recent study, C-F bond cleavage by an engineered enzyme has been reported [41]. In this study, cysteine dioxygenase is engineered by replacing the tyrosine residue at 157th by 3,5-difluoro-tyrosine (Fig.…”

“…9a). Cysteine dioxygenase has a protein-derived cysteine-tyrosine cofactor (C-S thioester bond between Cys93 and Tyr157 in human) in the enzyme itself [41,42]. The C-S thioester bond is formed by post-translational modification.…”

“…The engineered cysteine dioxygenase catalyzed the defluorination of its own 3,5-difluoro-tyrosine residue at 157th during cofactor formation (Fig. 9b) [41]. The C-F bonds have the highest dissociation energy found in organic compounds, which results in no ready breakdown of the substance containing the C-F bond in natural environments.…”

Enzyme-catalyzed C–F bond formation and cleavage

“…2; Tiedt et al 2016). In 2018, Liu et al reported cleavage of a C-F bond by an engineered cysteine dioxygenase ( Whittaker 2003;Simmons et al 2006;Li et al 2018). Experimental data suggest that protein-bound O 2 -dependent carbonhalogen bonds cleave iron centers under mild, physiologically relevant conditions.…”