Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

Abstract

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity

“…S8). Under the conditions tested therefore the unusual posttranslational modification observed to enhance turnover in mammalian CDOs was not identified in the AtPCOs, though we cannot rule out that a cross-link may be observable using different techniques (31).…”

“…This suggests that PCO enzymes cannot form an equivalent thioether cross-link; however, residues elsewhere in the AtPCO active sites do have the potential to form a similar posttranslational modification. Thioether crosslink formation has been observed in the active site of human cysteamine dioxygenase, HsADO, between Cys220 and Tyr222 (13,31). HsADO is a thiol dioxygenase which also catalyses oxidation of protein N-terminal cysteine residues and retains Fig.…”

“…Furthermore, AtPCO Asp176 is conserved both in PCOs from other plants and HsADO, supporting an important role for this residue in catalysis as evidenced by our mutagenesis experiments. Interestingly a Cys-Tyr thioether cross-link, a feature important in RnCDO and other small thiol dioxygenases (24,25,35), was detected in HsADO between Cys220 and Tyr222 following incubation with cysteamine (31). These residues are conserved in PCOs (AtPCO4 Cys190 and Tyr192, SI Appendix, Fig.…”

Structures, Spectroscopic Properties, and Dioxygen Reactivity of 5- and 6-Coordinate Nonheme Iron(II) Complexes: A Combined Enzyme/Model Study of Thiol Dioxygenases

“…It is plausible that the other thiol dioxygenases exhibit similar substrate-bound structures, although they have not yet been characterized by XRD. 2–3, 5…”

Emerging roles for thiol dioxygenases as oxygen sensors

“…Indeed as for CDO, iron coordination is needed for ADO activity and when iron binding was ablated using a His to Ala variant cysteamine dioxygenase activity was lost [35]. It is also possible that ADO shares another structural feature with CDO: in 2018, Wang et al published evidence that a Cys-Tyr crosslink can occur in ADO between Cys 220 and Tyr 222 [37]. Interestingly, recent spectroscopic investigations indicate that metal -substrate coordination in ADO is distinctive from that of CDO: While crystallographic evidence has shown these enzymes to bind their substrates in a bidentate manner, spectroscopic evidence has indicated that ADO binds cysteamine in a monodentate manner via the thiol but not via its amine group [38,39].…”