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:Z nucleobase 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" Z nucleobase. 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.

中文翻译：

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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。

获得利用“八字”DNA 信息密度的半合成微生物需要使用工程 DNA 聚合酶。据报道，KlenTaq 变体掺入了“hachimoji”P:Z 核碱基对，其效率与野生型 (WT) KlenTaq DNA 聚合酶的 Watson-Crick 核碱基掺入效率相似。变体聚合酶与 WT KlenTaq 的不同之处仅在于四个氨基酸取代，其中没有一个位于活性位点内。我们现在报告了一系列二元复合物的分子动力学（MD）模拟，旨在阐明四种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 AEGIS DNA，导致定位二元复合物以有效掺入“hachimoji”Z 核碱基所需的关键蛋白质/DNA 相互作用丢失。此外，我们测试了有关每个氨基酸取代的功能作用的文献假设，并提供了单个残基变化如何有助于提高 KlenTaq 变体活性的分子描述。我们证明，MD 模拟在系统筛选能够掺入不同类型非天然核碱基的 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表征的数量。现在可以构建除 A:T 和 G:C 之外还包含非天然核碱基对的 DNA 分子。利用合成生物学的这一发展需要能够复制非天然核碱基对的工程 DNA 聚合酶。对 DNA 聚合酶变体的计算研究揭示了活性位点之外的氨基酸取代如何产生一种能够高效复制非天然核碱基对的酶。这项工作将有助于获得具有扩展遗传字母表的细菌。