Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan; PREST, Japan Science and Technology Agency, Kawaguchi, Japan.
J Biol Chem. 2021 Sep;297(3):101043. doi: 10.1016/j.jbc.2021.101043. Epub 2021 Aug 4.
A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the k/K value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.
大量的蛋白质序列被注册在公共数据库中，如 PubMed。这些数据库中包含功能尚未明确的酶，其中一些可能具有工业应用的潜力。然而，目前酶的分类仍然是一项艰巨的任务。在这项研究中，我们将总共 28 个原始序列分配给了一些细菌物种中表达的 FAD 依赖氧化酶家族中的未明确酶，这些细菌包括黄杆菌属、噬纤维菌属和Pedobacter。分配的 28 个序列的祖先序列通过祖先序列重建生成，生成的序列表现出 L-赖氨酸氧化酶活性；因此，我们将该酶命名为 AncLLysO。AncLLysO 的配体自由和配体结合形式的晶体结构被确定，表明该酶通过与 R76 和 E383 形成氢键识别 L-Lys。L-Lys 与 AncLLysO 的结合诱导由残基 251 至 254 形成的塞子环的动态结构变化。AncLLysO 变体的生化分析揭示了这些底物识别残基和塞子环的功能重要性。R76A 和 E383D 变体也被观察到失去活性，并且 G251P 和 Y253A 突变的 k/K 值比 AncLLysO 低约 800 至 1800 倍，尽管底物与突变残基之间存在间接相互作用。总之，我们的数据表明，从数据库和祖先序列重建的组合序列分类方法不仅可以有效地利用未知序列的数据库来发现新的酶，还可以阐明它们的功能。
