Yashima Yuki, Takeda Kota, Sunagawa Naoki, Uchiyama Taku, Igarashi Kiyohiko
1 Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo.
2 National Institute of Advanced Industrial Science and Technology.
J Appl Glycosci (1999). 2025 May 20;72(2):7202108. doi: 10.5458/jag.7202108. eCollection 2025.
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 (POx), 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 POx were expected. Here, we explored the potential for amino acids other than histidine to covalently bind FAD in POx 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.
酶与辅因子的相互作用在酶功能中起着重要作用。特别是，蛋白质与黄素辅因子之间的共价键对于共价黄素蛋白中的酶活性和氧化还原电位很重要。例如，在担子菌的吡喃糖2-氧化酶（POx）中，黄素腺嘌呤二核苷酸（FAD）辅因子与组氨酸（His158）形成共价键，而其他黄素蛋白中的FAD可以与其他氨基酸残基形成共价键，如半胱氨酸、酪氨酸和天冬氨酸。考虑到与FAD形成共价键的机制，预计POx中会出现新的共价FAD模式。在这里，我们通过对His158进行全面的定点诱变，探索了除组氨酸之外的氨基酸与POx中的FAD共价结合的可能性，并评估了19个突变体与FAD形成共价键的能力，以及对D-葡萄糖的氧化酶和脱氢酶活性。所有突变体都未能与FAD形成共价键，尽管它们可以在不同程度上非共价结合FAD，但H158D和H158P除外，它们不仅失去了与FAD的共价键，还失去了整个FAD辅因子。与野生型酶相比，His158变体对D-葡萄糖的氧化酶和脱氢酶活性均显著降低。此外，脱辅基酶H158D和H158P无活性。我们的研究结果有望有助于黄素蛋白人工辅因子的设计。
