6XLR
The 1.23 Angstrom crystal structure of galactose oxidase variant with genetically incorporated Cl2-Tyr272
Summary for 6XLR
| Entry DOI | 10.2210/pdb6xlr/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 | 71317.74 |
| Authors | |
| 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 Cited by 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: 33091303DOI: 10.1021/jacs.0c08992 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.23 Å) |
Structure validation
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