Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. Structure–function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

中文翻译：

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来自Pycnoporus cinnabarinus的寡糖脱氢酶的晶体结构和功能表征为木质纤维素的真菌分解提供了见解

真菌葡萄糖脱氢酶 (GDH) 是属于葡萄糖-甲醇-胆碱氧化还原酶超家族的 FAD 依赖性酶。这些酶被归类在碳水化合物活性酶数据库的“辅助活性”家族 3 (AA3) 中，更具体地说，属于亚家族 AA3_2，其中还包括密切相关的黄素酶芳基醇氧化酶和葡萄糖 1-氧化酶。基于与已知真菌 GDH 的序列相似性，在 Pycnoporus cinnabarinus（一种能够完全降解木质素的担子菌模型）的基因组中鉴定出一种对葡萄糖有活性的 AA3_2 酶。在我们的工作中，底物筛选和功能表征显示该酶对含有 β(1→3) 糖苷键的寡糖具有出乎意料的优先活性，对二糖海带二糖观察到的效率最高。尽管其序列与 GDH 相似，但我们为该酶定义了一种新的酶活性，即寡糖脱氢酶 (ODH)。无糖形式以及与葡萄糖和海带二糖复合的 ODH 的晶体结构揭示了一种独特的糖识别机制，该机制与先前表征的 AA3 氧化还原酶不同，并且解释了 ODH 对寡糖的优先活性。ODH 活性位点中的糖分子主要通过 CH-π 与芳香族残基的相互作用来稳定，而不是通过与高度保守的残基的氢键来稳定，正如迄今为止所观察到的真菌葡萄糖脱氢酶和氧化酶所观察到的那样。最后，在 ODH 外表面鉴定了三个糖结合位点，这是以前没有观察到的，可能在生理场景中很重要。ODH 的结构-功能分析与其在木质纤维素降解中作为辅助酶的作用一致，并揭示了碳水化合物活性酶数据库的 AA3 家族中的另一种酶功能。我们的研究结果可以破译底物结合的分子决定因素，并提供对 ODH 生理作用的洞察，为利用生物多样性将木质纤维素转化为燃料和化学品开辟了新的视角。