Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Research Center for Functional Materials, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki, 305-0047, Japan.
Bioelectrochemistry. 2018 Aug;122:115-122. doi: 10.1016/j.bioelechem.2018.03.001. Epub 2018 Mar 7.
Glucoside 3‑dehydrogenase (G3DH) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase that catalyzes the oxidation of the hydroxy group on the C-3 position of pyranose and shows broad substrate specificity by oxidizing many saccharides. Due to unique site specificity and wide substrate specificity, G3DHs can be used for synthesis of sugar derivatives, anodic catalysis in biofuel cells, multi-sugar analysis using enzyme electrode, and for enzymatic detection of 1,5‑anhydro‑d‑glucitol, a clinical marker for diabetes. However, few studies have focused on the fundamental biochemical properties of G3DH, including its electron transfer pathway. In this study, we isolated the G3DH gene from Rhizobium radiobacter, a homologue of marine bacterial G3DH, and reported that the isolated gene fragment contains the genes encoding the G3DH catalytic subunit (subunit I), G3DH hitch-hiker subunit (subunit II), and cytochrome c-like molecule (CYTc). Furthermore, we report the recombinant expression of G3DH from R. radiobacter in Escherichia coli, the characterization of recombinant G3DH and the investigation of the molecular electron pathway of G3DH. We first prepared the G3DH subunit I-II complex using a co-expression vector for both subunits. The G3DH subunit I-II complex showed dye-mediated G3DH activity toward methyl‑α‑d‑glucoside (MαG). Electron paramagnetic resonance (EPR) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses revealed that subunit I contains an iron-sulfur cluster. We, then, prepared recombinant CYTc and revealed that it is capable of accepting electrons from the catalytic subunit of G3DH by absorption spectrum analysis. These results suggested that R. radiobacter G3DH possesses an iron‑sulfur cluster that may play an important role in the electron transfer from FAD to cytochrome c like molecule, which is an external electron acceptor of G3DH. Furthermore, we demonstrated that CYTc mediate the electron transfer from G3DH to electrode without the artificial electron mediator.
葡萄糖苷 3-脱氢酶 (G3DH) 是一种黄素腺嘌呤二核苷酸 (FAD) 结合氧化还原酶，可催化吡喃糖 C-3 位上的羟基氧化，通过氧化许多糖显示出广泛的底物特异性。由于独特的位点特异性和广泛的底物特异性，G3DH 可用于糖衍生物的合成、生物燃料电池中的阳极催化、使用酶电极的多糖分析以及 1,5-脱水-D-葡萄糖醇的酶促检测，1,5-脱水-D-葡萄糖醇是糖尿病的临床标志物。然而，很少有研究关注 G3DH 的基本生化特性，包括其电子转移途径。在这项研究中，我们从海洋细菌 G3DH 的同源物根瘤菌中分离出 G3DH 基因，并报告说分离的基因片段包含编码 G3DH 催化亚基（亚基 I）、G3DH 搭便车亚基（亚基 II）和细胞色素 c 样分子（CYTc）的基因。此外，我们报告了来自 R. radiobacter 的 G3DH 在大肠杆菌中的重组表达、重组 G3DH 的表征以及 G3DH 分子电子途径的研究。我们首先使用两个亚基的共表达载体制备 G3DH 亚基 I-II 复合物。G3DH 亚基 I-II 复合物显示出对甲基-α-D-吡喃葡萄糖苷 (MαG) 的染料介导 G3DH 活性。电子顺磁共振 (EPR) 和电感耦合等离子体发射光谱 (ICP-OES) 分析表明，亚基 I 包含一个铁硫簇。然后，我们制备了重组 CYTc，并通过吸收光谱分析表明它能够从 G3DH 的催化亚基接受电子。这些结果表明，R. radiobacter G3DH 具有铁硫簇，它可能在 FAD 向细胞色素 c 样分子的电子转移中发挥重要作用，细胞色素 c 样分子是 G3DH 的外部电子受体。此外，我们证明 CYTc 在没有人工电子介体的情况下介导 G3DH 向电极的电子转移。
