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The 1.80 Angstrom crystal structure of galactose oxidase variant with genetically incorporated F2-Tyr272
Structural highlights
FunctionGAOA_GIBZA Catalyzes the sterospecific oxidation of primary alcohols to the corresponding aldehydes. The biologically relevant substrate of the enzyme is not known as the enzyme exhibits broad substrate specificity from small alcohols through sugars to oligo- and polysaccharides.[1] [2] Publication Abstract from PubMedGalactose 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 (Cl2-Tyr) and 3,5-difluorotyrosine (F2-Tyr) to replace Tyr272 in the GAO(V) 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(V), Cl2-Tyr272, and F2-Tyr272 incorporated variants at 1.48, 1.23, and 1.80 A 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. Formation of Monofluorinated Radical Cofactor in Galactose Oxidase through Copper-Mediated C-F Bond Scission.,Li J, Davis I, Griffith WP, Liu A J Am Chem Soc. 2020 Nov 4;142(44):18753-18757. doi: 10.1021/jacs.0c08992. Epub, 2020 Oct 22. PMID:33091303[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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