Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers
Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers
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DOI：
10.1093/protein/gzx003
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发表时间：
2017-04-01
影响因子：
2.4
通讯作者：
Tawfik, Dan S.
中科院分区：
文献类型：
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作者：
Goldsmith, Moshe;Aggarwal, Nidhi;Tawfik, Dan S.
将酶最初对新目标底物的低催化效率提高一两个数量级，可能只需要几轮诱变和筛选或选择。然而，随后的几轮优化往往会产生越来越少的改进程度（收益递减），最终导致优化平台期。我们旨在优化细菌磷酸三酯酶（PTE）对v型神经毒剂的催化效率。之前，我们将野生型PTE对神经毒剂VX的催化效率提高了500倍，达到催化效率（k(cat)/ k -M）为5 × 10(6)M(-1) min（-1）。然而，有效的体内解毒需要一种催化效率为bbb10 (7) M-1 min（-1）的酶。在这里，经过额外的8轮定向进化和稳定变体的计算设计，我们进化出PTE变体，其解毒VX的k(cat)/ k -M >= 5 × 10(7)M(-1) min（-1）和俄罗斯VX （RVX）的k(cat)/ k -M >= 10(7)M -1 min（-1）。这些最后的10倍改进是最耗时和费力的，因为大多数库要么只产生很小的改进，要么没有。稳定进化中的酶，避免与不同底物的活性权衡，使我们能够在优化平台之外获得进一步的改进，并进化出PTE变体，VX和RVX的PTE变体总体上分别提高了> 5000倍和> 17000倍。由此产生的变体还能高效水解g型神经毒剂(GA， GB在k(cat)/ k - m > 5 × 10(7) M-1 min(-1))，因此可以作为广谱神经毒剂预防和低酶剂量暴露后治疗的候选物。
Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (k(cat)/K-M) of 5 x 10(6)M(-1) min(-1). However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of > 10(7) M-1 min(-1). Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a k(cat)/K-M >= 5 x 10(7)M(-1) min(-1) and Russian VX (RVX) with a k(cat)/K-M >= 10(7) M-1 min(-1). These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by > 5000-fold with VX and by > 17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at k(cat)/K-M > 5 x 10(7) M-1 min(-1)) and can thus serve as candidates for broadspectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.