Doubayashi Daiju, Ootake Takumi, Maeda Yosifumi, Oki Masaya, Tokunaga Yuji, Sakurai Akihiko, Nagaosa Yukio, Mikami Bunzo, Uchida Hiroyuki
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui.
Biosci Biotechnol Biochem. 2011;75(9):1662-7. doi: 10.1271/bbb.110153. Epub 2011 Sep 7.
Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be -47 and -177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His₅₁₁ and Arg₅₅₄ were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.
米曲霉RIB40的甲酸氧化酶含有一种分子量为799的8-取代FAD作为辅因子。从该酶获得的辅因子部分的¹H-NMR光谱表明，该部分中的8-取代FAD为8-甲酰基-FAD，以开放形式和半缩醛形式存在。通过循环伏安法估计，开放形式和半缩醛形式的氧化还原电位相对于标准氢电极分别为-47和-177 mV。利用从该酶晶体收集的分辨率为2.24 Å的衍射数据构建了该酶的结构。His₅₁₁和Arg₅₅₄位于开放形式的8-甲酰基-FAD异咯嗪环嘧啶部分附近。该酶含有8-甲酰基-FAD，其氧化电位比FAD的氧化电位约正160 mV，并且在催化位点存在His-Arg对，这与属于葡萄糖-甲醇-胆碱氧化还原酶家族的其他酶不同。
