Designing Allosteric Control into Enzymes by Chemical Rescue of Structure
Designing Allosteric Control into Enzymes by Chemical Rescue of Structure
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DOI：
10.1021/ja301409g
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发表时间：
2012-06-20
影响因子：
15
通讯作者：
Karanicolas, John
中科院分区：
文献类型：
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作者：
Deckert, Katelyn;Budiardjo, S. Jimmy;Karanicolas, John
配体依赖性活性已被设计到酶中，其用途包括从控制细胞形态到重新编程细胞信号传导途径。这些成功通常将天然变构域融合到感兴趣的酶中，而在这里我们展示了一种将从头变构效应位点直接设计到酶催化域中的方法。这种方法与传统的酶化学拯救不同，因为它依赖于结构的破坏和恢复，而不是活性位点化学，作为实现调节功能的手段。我们提供了两个例子，β-糖苷酶 (β-gly) 中的 W33G 和 β-葡萄糖醛酸酶 (β-gluc) 中的 W492G，其中我们通过去除作为活性位点结构支撑的埋藏色氨酸侧链，将吲哚依赖性活性设计到酶中。在这两种情况下，我们都观察到功能丧失，并且在这两种情况下，我们发现随后添加吲哚可用于恢复活性。通过对 β-gly W33G 动力学的详细分析，我们证明这种被拯救的酶在功能上完全等同于相应的野生型酶。然后，我们展示了 β-gly W33G 的 apo 和吲哚结合晶体结构，它们共同建立了酶失活和拯救的结构基础。最后，我们使用这种设计的开关通过吲哚调节活细胞中的 β-糖苷酶活性。蛋白质结构的破坏和恢复可能代表了将变构控制引入酶的通用技术，因此可以作为构建各种生物开关和传感器的起点。
Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a beta-glycosidase enzyme (beta-gly) and W492G in a beta-glucuronidase enzyme (beta-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of beta-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of beta-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate beta-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.