6XLT

The 1.48 Angstrom crystal structure of evolved galactose oxidase variant A3.E7

Summary for 6XLT

• Entry DOI: 10.2210/pdb6xlt/pdb
• Descriptor: Galactose oxidase, COPPER (II) ION, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: cys-tyr cofactor, oxidoreductase
• Biological source: Gibberella zeae (Wheat head blight fungus)
• Total number of polymer chains: 1
• Total formula weight: 70117.73

Authors

Liu, A.,Li, J. (deposition date: 2020-06-29, release date: 2021-02-03, Last modification date: 2023-10-18)

Primary citation

Li, J.,Davis, I.,Griffith, W.P.,Liu, A.
Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C-F Bond Scission.
J.Am.Chem.Soc., 142:18753-18757, 2020

PubMed Abstract: Galactose oxidase (GAO) contains a Cu(II)-ligand radical cofactor. The cofactor, which is autocatalytically generated through the oxidation of the copper, consists of a cysteine-tyrosine radical (Cys-Tyr) as a copper ligand. The formation of the cross-linked thioether bond is accompanied by a C-H bond scission on Tyr272 with few details known thus far. Here, we report the genetic incorporation of 3,5-dichlorotyrosine (Cl-Tyr) and 3,5-difluorotyrosine (F-Tyr) to replace Tyr272 in the GAO previously optimized for expression through directed evolution. The proteins with an unnatural tyrosine residue are catalytically competent. We determined the high-resolution crystal structures of the GAO, Cl-Tyr272, and F-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 Å resolution, respectively. The structural data showed only one halogen remained in the cofactor, indicating that an oxidative carbon-chlorine/fluorine bond scission has occurred during the autocatalytic process of cofactor biogenesis. Using hydroxyurea as a radical scavenger, the spin-coupled hidden Cu(II) was observed by EPR spectroscopy. Thus, the structurally defined catalytic center with genetic unnatural tyrosine substitution is in the radical containing form as in the wild-type, i.e., Cu(II)-(Cl-Tyr-Cys) or Cu(II)-(F-Tyr-Cys). These findings illustrate a previously unobserved C-F/C-Cl bond cleavage in biology mediated by a mononuclear copper center.

PubMed: 33091303
DOI: 10.1021/jacs.0c08992

Experimental method

X-RAY DIFFRACTION (1.478 Å)