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首页> 外文期刊>Journal of Molecular Biology >X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism
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X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

机译：pH 4.5时绿球菌乳酸氧化酶及其与D-乳酸的复合物的X射线结构显示出α-羟酸的氧化机理

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摘要

L-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent alpha-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with D-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the D-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the D-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between D-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O-2 could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes. (c) 2008 Elsevier Ltd. All rights reserved.
机译：L-乳酸氧化酶（LOX）属于黄素单核苷酸（FMN）依赖性α-羟酸氧化酶家族。以前，已解决了来自绿球菌的LOX（pH 8.0）的晶体结构，发现活性位点残基位于FMN周围。在这里，我们分析了相同酶在pH 4.5的晶体结构及其与D-乳酸在pH 4.5的配合物，以试图分析中间步骤。在复杂的结构中，D-乳酸存在于底物结合位点，但有趣的是，与其在pH 8.0的未结合状态构象相比，活性位点His265远离D-乳酸翻转。该运动可能是由于在pH 4.5的结晶过程中His265的质子化所致，因为在pH 4.5的未结合状态的结构中观察到了相同的翻转。因此，在His265从底物提取质子之后，本结构似乎模仿了中间体。 His265的翻转触发了大的结构重排，在His265-Asp174-Lys221之间创建了一个新的氢键网络，并且进一步在D-乳酸和His265之间引入了分子氧。三元复合中间酶-底物-O-2的模拟可以解释通过氢化物转移释放丙酮酸的还原半反应机理。在随后的氧化半反应的机理中，His265向后翻转，将分子氧推入作为第二种底物的底物结合位点，然后发生逆反应生成过氧化氢。在反应过程中，His265的触发器作用具有双重作用，可作为活性碱/酸来定义主要的化学步骤。我们提出的反应机制似乎是该酶家族的常见机制。 （c）2008 Elsevier Ltd.保留所有权利。

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