Zhang Lan, Laurent Christophe V F P, Schwaiger Lorenz, Wang Lushan, Ma Su, Ludwig Roland
Department of Food Science and Technology, Biocatalysis and Biosensing Laboratory, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, Vienna 1190, Austria.
Institute of Molecular Modeling and Simulation, Department of Material Sciences and Process Engineering, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, Vienna 1190, Austria.
ACS Catal. 2023 Jun 5;13(12):8195-8205. doi: 10.1021/acscatal.3c02116. eCollection 2023 Jun 16.
Direct bioelectrocatalysis applied in biosensors, biofuel cells, and bioelectrosynthesis is based on an efficient electron transfer between enzymes and electrodes in the absence of redox mediators. Some oxidoreductases are capable of direct electron transfer (DET), while others achieve the enzyme to electrode electron transfer (ET) by employing an electron-transferring domain. Cellobiose dehydrogenase (CDH) is the most-studied multidomain bioelectrocatalyst and features a catalytic flavodehydrogenase domain and a mobile, electron-transferring cytochrome domain connected by a flexible linker. The ET to the physiological redox partner lytic polysaccharide monooxygenase or, ex vivo, electrodes depends on the flexibility of the electron transferring domain and its connecting linker, but the regulatory mechanism is little understood. Studying the linker sequences of currently characterized CDH classes we observed that the inner, mobile linker sequence is flanked by two outer linker regions that are in close contact with the adjacent domain. A function-based definition of the linker region in CDH is proposed and has been verified by rationally designed variants of CDH. The effect of linker length and its domain attachment on electron transfer rates has been determined by biochemical and electrochemical methods, while distances between the domains of CDH variants were computed. This study elucidates the regulatory mechanism of the interdomain linker on electron transfer by determining the minimum linker length, observing the effects of elongated linkers, and testing the covalent stabilization of a linker part to the flavodehydrogenase domain. The evolutionary guided, rational design of the interdomain linker provides a strategy to optimize electron transfer rates in multidomain enzymes and maximize their bioelectrocatalytic performance.
应用于生物传感器、生物燃料电池和生物电合成的直接生物电催化基于在没有氧化还原介质的情况下酶与电极之间的高效电子转移。一些氧化还原酶能够进行直接电子转移（DET），而其他氧化还原酶则通过采用电子转移结构域来实现酶到电极的电子转移（ET）。纤维二糖脱氢酶（CDH）是研究最多的多结构域生物电催化剂，其特征在于一个催化黄素脱氢酶结构域和一个通过柔性接头连接的可移动电子转移细胞色素结构域。向生理氧化还原伙伴裂解多糖单加氧酶或离体电极的电子转移取决于电子转移结构域及其连接接头的灵活性，但调控机制鲜为人知。通过研究当前已表征的CDH类别的接头序列，我们观察到内部可移动接头序列两侧是与相邻结构域紧密接触的两个外部接头区域。本文提出了基于功能的CDH接头区域定义，并通过合理设计的CDH变体进行了验证。接头长度及其与结构域的连接对电子转移速率的影响已通过生化和电化学方法确定，同时计算了CDH变体结构域之间的距离。本研究通过确定最小接头长度、观察延长接头的影响以及测试接头部分与黄素脱氢酶结构域的共价稳定性，阐明了结构域间接头对电子转移的调控机制。基于进化指导的结构域间接头合理设计提供了一种优化多结构域酶中电子转移速率并最大化其生物电催化性能的策略。
