Building better enzymes: Molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
Building better enzymes: Molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.
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构建更好的酶：通过进化的 DNA 聚合酶改进非天然核碱基掺入的分子基础。
DOI：
10.1002/pro.3762
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Richards,NigelGJ
中科院分区：
文献类型：
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作者：
Ouaray,Zahra;Singh,Isha;Georgiadis,MillieM;Richards,NigelGJ
要获得利用“hachimoji”DNA信息密度的半合成微生物，需要获得工程DNA聚合酶。据报道，一种KlenTaq变体整合了“hachimoji”P：Z核苷酸碱基对，其效率与野生型(WT)KlenTaq DNA聚合酶掺入Watson-Crick核苷酸碱基的效率相似。变异型聚合酶与WT KlenTaq只有四个氨基酸替换，没有一个位于活性部位。我们现在报道一系列二元络合物的分子动力学(MD)模拟，旨在阐明四个氨基酸取代对改变催化活性的贡献。这些模拟表明，WT KlenTaq不够灵活，无法正确结合宙斯盾DNA，导致失去了定位二元复合体所需的关键蛋白质/DNA相互作用，从而有效地掺入了“hachimoji”Zna碱基。此外，我们检验了有关每个氨基酸替代的功能作用的文献假设，并提供了单个残基的变化如何有助于KlenTaq变体活性提高的分子描述。我们证明，MD模拟在系统地筛选能够整合不同类型的非天然核苷酸碱基的DNA聚合酶变体方面具有明显的作用，从而限制了需要通过实验来表征的数量。现在，除了A：T和G：C之外，还可以构建含有非自然碱基对的DNA分子。要在合成生物学中利用这一发展，需要能够复制非自然碱基对的工程DNA聚合酶。对DNA聚合酶变异体的计算研究揭示了活性部位外的氨基酸替代如何产生一种高效复制非天然核碱基对的酶。这项工作将促进获得拥有扩展基因字母表的细菌的努力。
Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji”P:Znucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji”Znucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.