Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases
Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases
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爪臂状环的赖氨酸突变加速纤维二糖水解酶的催化
DOI：
10.1021/jacs.9b08477
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发表时间：
2019
影响因子：
15
通讯作者：
Shao Xueguang
中科院分区：
文献类型：
--
作者：
Zong Zhiyou;Li Qiyu;Hong Zhangyong;Fu Haohao;Cai Wensheng;Chipot Christophe;Jiang Huifeng;Zhang Dongyuan;Chen Shulin;Shao Xueguang
为了寻找加速糖苷水解酶家族7（GH 7）纤维二糖水解酶I（CBHI）催化循环的可行策略，我们对GH 7 CBHI进行了总共12 μs的分子动力学模拟，揭示了一种新的机制。纤维二糖排出的机制，被称为“爪臂”作用。产物结合位点侧翼的环起着向纤维二糖延伸的柔性“臂”的作用，并且该环的残基Thr 389充当捕获纤维二糖的“爪”。残基Thr 389的五个突变被认为增强了环-纤维二糖相互作用。赖氨酸突变体被发现显着加速纤维二糖驱逐和促进多糖链易位。埃默森篮状菌CBHI（TeCel 7A）中Thr 393的赖氨酸突变表现类似。赖氨酸接近催化区域并稳定米氏复合物，可能影响糖基化，即催化循环的限速步骤。QM/MM计算表明，赖氨酸取代使质子转移（糖基化的关键步骤）的屏障降低了2.3 kcal/mol。使用全长野生型（WT）ofTeCel 7A及其突变体（在巴斯德毕赤酵母中重组表达）进行实验验证以降解底物。与野生型相比，赖氨酸突变体揭示了相关的更高的酶促反应速率。此外，纤维二糖产量也增加了赖氨酸突变，表明从纤维素的酶的解离加速，这在很大程度上源于增强的灵活性的“臂”。目前的工作设想，以帮助设计策略，提高酶的活性，同时降低酶的成本。
Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI)—the workhorse cellulose-degrading enzymes, we have performed a total of 12-μs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined “claw-arm” action. The loop flanking the product binding site plays the role of a flexible “arm” extending toward cellobiose, and residue Thr389 of this loop acts as a “claw” that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to significantly accelerate cellobiose expulsion and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 inTalaromyces emersoniiCBHI (TeCel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) ofTeCel7A and its mutants, recombinantly expressed inPichia pastoris, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the “arm”. The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.