Comprehensive Analysis of Histidine158 Variants Reveals Histidine Is Essential for Covalent Flavin Attachment in Pyranose 2-Oxidase

Abstract

Enzymes and cofactors interactions play a significant role in enzymatic function. Particularly, the covalent bonds between proteins and flavin cofactors are important for enzymatic activity and redox potential in covalent flavoproteins. For example, in pyranose 2-oxidase from the basidiomycete Phanerochaete chrysosporium (PcPOx), the flavin adenine dinucleotide (FAD) cofactor forms a covalent bond with histidine (His158), while FAD in other flavoproteins can form a covalent bond with other amino acid residues, such as cysteine, tyrosine, and aspartic acid. Considering the mechanism of forming a covalent bond with FAD, new covalent FAD patterns in PcPOx were expected. Here, we explored the potential for amino acids other than histidine to covalently bind FAD in PcPOx by conducting comprehensive site-directed mutagenesis at His158, and evaluated 19 mutants for covalent-bond-forming ability with FAD, as well as for oxidase and dehydrogenase activities towards D-glucose. All the mutants failed to form a covalent bond with FAD, though they could bind FAD noncovalently to various extents, except for H158D and H158P, which lost not only the covalent bonds with FAD but also the whole of FAD cofactors. The His158 variants showed markedly reduced both the oxidase and dehydrogenase activity toward D-glucose compared with the wild-type enzyme. Moreover, the apo-enzymes H158D and H158P were inactive. Our findings are expected to be helpful in the design of artificial cofactors for flavoproteins.