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.

葡糖苷3-脱氢酶（G3DH）是一种含有黄素腺嘌呤二核苷酸（FAD）的氧化还原酶，可催化吡喃糖C-3位置上的羟基氧化，并通过氧化许多糖而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，G3DHs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用于酶法检测1,5-脱水d-葡萄糖醇（一种糖尿病的临床标志。但是，很少有研究集中在G3DH的基本生化特性上，包括其电子转移途径。在这项研究中，我们从根瘤菌中分离了G3DH基因，是海洋细菌G3DH的同源物，并报道了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，我们报道了来自R. radiobacter的G3DH在大肠杆菌中的重组表达，重组G3DH的表征以及G3DH的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了G3DH亚基I-II复合物。G3DH亚基I-II复合物显示出染料介导的G3DH对甲基-α- d的活性葡糖苷（MαG）。电子顺磁共振（EPR）和电感耦合等离子体发射光谱（ICP-OES）分析显示，亚基I包含铁硫簇。然后，我们制备了重组CYTc，并通过吸收光谱分析揭示了它能够接受来自G3DH催化亚基的电子。这些结果表明，R.放射形G3DH具有铁-硫簇，其可以在从FAD的电子转移到细胞色素发挥重要作用Ç样分子，其是G3DH的外部电子受体。此外，我们证明了CYTc介导了从G3DH到电极的电子转移，而没有人工电子介体。