A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase
A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase
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DOI：
10.1111/j.1742-4658.2009.07222.x
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发表时间：
2009-09-01
期刊：
影响因子：
5.4
通讯作者：
Macheroux, Peter
中科院分区：
文献类型：
--
作者：
Binter, Alexandra;Staunig, Nicole;Macheroux, Peter
YhdA是一种来自枯草芽孢杆菌的热稳定NADPH：FMN氧化还原酶，通过乒乓双双机制还原醌类，对NADPH具有明显的偏好。该酶在溶液中以稳定的四聚体形式存在。两个延伸的二聚体表面通过一个二聚体相对于另一个二聚体旋转90度而彼此压紧。该组装通过在相邻原聚体的K109和D137之间形成四个盐桥来稳定。为了研究离子对接触的重要性，产生K109 L和D137 L单置换变体，以及K109 L/D137 L和K109 D/D137 K双置换变体，表达，纯化，结晶和生物化学表征。K109 L和D137 L变体形成二聚体而不是四聚体，而K109 L/D137 L和K109 D/D137 K变体似乎在溶液中以二聚体-四聚体平衡存在。K109 L和D137 L变体的晶体结构证实了二聚体状态，其中K109 L/D137 L和K109 D/D137 K变体采用四聚体组装。有趣的是，所有蛋白质变体在稳态动力学中显示出显著降低的醌还原酶活性。两个半反应的详细分析表明，氧化半反应不受影响，而还原的结合FMN辅因子的NADPH几乎被废除。对晶体结构的检查表明，K109的侧链通过形成与D137的盐桥以及稳定FMN辅因子附近的富含甘氨酸的环而发挥双重作用。在所有蛋白质变体中，与野生型相比，这种富含甘氨酸的环表现出更高的迁移率。这似乎与NADPH结合不相容，因此导致黄素还原的废除。
YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90 degrees rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137 of the neighbouring protomers. To investigate the importance of the ion pair contacts, the K109L and D137L single replacement variants, as well as the K109L/D137L and K109D/D137K double replacement variants, were generated, expressed, purified, crystallized and biochemically characterized. The K109L and D137L variants form dimers instead of tetramers, whereas the K109L/D137L and K109D/D137K variants appear to exist in a dimer-tetramer equilibrium in solution. The crystal structures of the K109L and D137L variants confirm the dimeric state, with the K109L/D137L and K109D/D137K variants adopting a tetrameric assembly. Interestingly, all protein variants show a drastically reduced quinone reductase activity in steady-state kinetics. Detailed analysis of the two half reactions revealed that the oxidative half reaction is not affected, whereas reduction of the bound FMN cofactor by NADPH is virtually abolished. Inspection of the crystal structures indicates that the side chain of K109 plays a dual role by forming a salt bridge to D137, as well as stabilizing a glycine-rich loop in the vicinity of the FMN cofactor. In all protein variants, this glycine-rich loop exhibits a much higher mobility, compared to the wild-type. This appears to be incompatible with NADPH binding and thus leads to abrogation of flavin reduction.