Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign.
Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign.
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DOI：
10.1021/acschembio.2c00373
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发表时间：
2022-07-15
影响因子：
4
通讯作者：
Izgu, Enver Cagri
中科院分区：
文献类型：
--
作者：
Aggarwal, Tushar;Hansen, William A.;Hong, Jonathan;Ganguly, Abir;York, Darrin M.;Khare, Sagar D.;Izgu, Enver Cagri
DNA聚合酶已经进化到在生命树中具有高度保守的活性：无一例外地形成核苷酸间O-P键。这种选择性连接是否可以通过设计来克服以产生异种核酸？在这里，我们报告了古细菌DNA聚合酶9°N的结构导向的重新设计，表现出一种在野生型酶中检测不到的新活性：使用3′-NH 2-ddNTPs催化核苷酸间N─P键的形成。用天冬酰胺取代9°N活性位点中的金属结合天冬氨酸是这种非自然酶活性出现的关键。MD模拟提供了对单个取代如何增强活性位点中3′-氨基亲核试剂的生产性定位的见解。指状亚结构域中蛋白质-核酸界面的进一步重构产生了显示DNA依赖性NP-DNA聚合酶活性的四重突变变体（9°N-NRQS）。此外，利用9°N-NRQS的工程化混杂性用于DNA-NP-DNA共聚物的一锅法合成。这项工作揭示了底物保真度和潜在的混杂酶的分子基础。
DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O─P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N─P linkages using 3′-NH2-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3′-amino nucleophile in the active site. Further remodeling of the protein–nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA—NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.
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影响因子：
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