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 (F2‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and 19F 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.

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

“…ADO is the sole gene product responsible for converting cysteamine to hypotaurine ( 13 ). Its presence constitutes an important step in the metabolic scheme of l -cysteine or coenzyme A to taurine and the thiol metabolism cycle in mammalian cells ( 14 ). ADO was initially thought of as a sibling enzyme of CDO but oxidizing cysteamine, which is the only reaction in the thiol dioxygenase family that involves a small-molecule substrate without a carboxylate moiety ( 2 , 13 ).…”

“…Its significance, however, has been increasingly appreciated since the recent discovery that ADO functions as an oxygen sensor involved in N -degron pathways ( 12 ). In our previous studies, we cloned and expressed human ADO (hADO) and characterized its spectroscopic and kinetic properties ( 14 , 21 ). We found that ADO can autocatalytically generate a Cys-Tyr cofactor ( 14 ).…”

“…In our previous studies, we cloned and expressed human ADO (hADO) and characterized its spectroscopic and kinetic properties ( 14 , 21 ). We found that ADO can autocatalytically generate a Cys-Tyr cofactor ( 14 ). The Cys-Tyr cofactor, cross-linked between Cys93 and Tyr157 through a thioether bond, was first identified in CDO in 2006 ( 5 , 6 ).…”

“…The Cys-Tyr cofactor, cross-linked between Cys93 and Tyr157 through a thioether bond, was first identified in CDO in 2006 ( 5 , 6 ). The cofactor of ADO is formed by two residues close in sequence (Cys220 and Tyr222) and is predicted to be located on a different side of the iron center ( 14 ). For both enzymes, mutation of either Cys or Tyr could lead to a failure of cross-link formation and decreased catalytic efficiency by at least an order of magnitude ( 14 , 22 ).…”

“…The cofactor of ADO is formed by two residues close in sequence (Cys220 and Tyr222) and is predicted to be located on a different side of the iron center ( 14 ). For both enzymes, mutation of either Cys or Tyr could lead to a failure of cross-link formation and decreased catalytic efficiency by at least an order of magnitude ( 14 , 22 ). The decreased activity, however, is not simply due to structural changes of the active site or the disruption of substrate binding but is associated with a critical role of the Cys-Tyr cofactor in catalysis.…”