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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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.
The authors declare no competing interests.
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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). https://doi.org/10.1038/s41589-018-0085-5