Explaining Operational Instability of Amine Transaminases: Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability
Explaining Operational Instability of Amine Transaminases: Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability
复制标题
DOI：
10.1021/acscatal.6b02100
复制
发表时间：
2017-02-01
期刊：
影响因子：
12.9
通讯作者：
Grey, Carl
中科院分区：
文献类型：
--
作者：
Borner, Tim;Ramisch, Sebastian;Grey, Carl
胺转氨酶（ATA）的操作稳定性不足构成了手性胺合成中高产率的限制因素。在这项工作中，我们研究了一个四聚ATA的操作稳定性与92%的序列同一性假单胞菌属转氨酶，并比较它的两个常用的二聚ATA从紫色色杆菌和河流弧菌。在底物的存在下，所有三种ATA的特征是与其静止稳定性相比稳定性降低，但四聚体显示出比二聚体ATA更慢的失活速率。动力学和热力学分析揭示了胺供体诱导的失活机制，涉及不太稳定的胺化酶辅因子中间体的积累。酶PMP复合物的解离形成不稳定的脱辅基酶，其可以迅速展开。晶体结构分析揭示了结构功能关系，表明辅因子环结合元件稳定在四级结构中，通过最大限度地减少PMP泄漏和脱辅基酶的形成，赋予更高的操作稳定性。与通常的做法相反，增加胺受体含量提高了二聚ATA的稳定性和底物周转率。吡哆醛辅因子（PLP）的额外供应增强了二聚体和四聚体ATA的稳定性，但降低了转氨活性。本文所述的ATA失活机制为工艺开发和蛋白质工程提供了有价值的方面。
The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme cofactor intermediate. Dissociation of the enzyme PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure function relationship suggesting that the cofactor ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.