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

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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 19F 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.

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Fig. 1: Crystal structures of human CDO and incorporation of unnatural amino acids into the catalytic active site tyrosine.
Fig. 2: MS/MS spectra of cross-linked peptides of WT and F2-Tyr157 CDO proteins and 19F NMR detection of leaving fluoride.
Fig. 3: Crystal structures of F2-Tyr157 CDO.
Fig. 4: Crystal structures of Cl-Tyr157 CDO.


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The work is supported in part by the National Institutes of Health grants GM107529, GM108988, and MH107985, the National Science Foundation grant CHE-1623856, and the Lutcher Brown Distinguished Chair Endowment fund (to A.L.). J.W. acknowledges the support of the National Science Foundation of China grants (91527302, 31370016, and U1532150). The mass spectrometry facility is sponsored by the National Institutes of Health grant G12MD007591. The MALDI-TOF and NMR spectrometers are shared instruments sponsored by the National Science Foundation under the award numbers #1126708 and 1625963, respectively. X-ray synchrotron data were collected at the beamlines of the Advanced Photon Source Section 19, Structural Biology Center user program GUP-48198, Argonne National Laboratory and at the beamline BL9-2 of the Stanford Synchrotron Radiation Lightsource (SSRL) under the user program #5B14, SLAC National Accelerator Laboratory. The beamline staff scientists are acknowledged for the assistance of the remote data collections. The Advanced Photon Source is a US Department of Energy, Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. SSRL is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515 and by the National Institutes of Health (P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Institutes of Health or National Science Foundation.

Author information

Genetic incorporation of unnatural amino acids was performed by J.L. (cloning, protein expression and purification, and enzyme assays). J.W. and F.L. provided TyrRS. W.P.G. and J.L. conducted mass spectrometry analyses. J.L. obtained all protein crystals, collected X-ray diffraction data, and interpreted and refined the structural data together with I.S. The mechanistic models were proposed and refined by J.L., I.D., Y.W., and A.L. Y.W. participated in the unnatural amino acid production and isolation by an enzymatic method. D.J.W. performed the 19F NMR analysis. A.L. conceived the research and wrote the manuscript together with J.L. All authors contributed to data analysis and to the writing and editing of the manuscript.

Correspondence to Aimin Liu.

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Li, J., Griffith, W.P., Davis, I. et al. Cleavage of a carbon–fluorine bond by an engineered cysteine dioxygenase. Nat Chem Biol 14, 853–860 (2018) doi:10.1038/s41589-018-0085-5

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