Abstract

Metallohydrolases are broadly used throughout biology, often to catalyze the degradation of macromolecules such as DNA and proteins. Many of these enzymes function with zinc in their active site, and an important subset of these enzymes utilize a binuclear zinc active site. Mimics of these enzymes have been developed, some of which catalyze the digestion of DNA. However, the majority of the mimics that utilize zinc are small molecules, and most are mononuclear. Herein, we report DNA cleavage activity by the de novo designed Due Ferri single-chain (DFsc) protein containing a binuclear zinc active site. This binuclear zinc–protein complex is able to digest plasmid DNA at rates up to 50 ng/h, and these cleavage rates are affected by changes to amino acid residues near the zinc-binding site. These results indicate that the DFsc scaffold is a good model system to carry out careful structure–function relationship studies to understand key structural features that influence reactivity in natural binuclear zinc hydrolases, as it is the first report of a binuclear model system in a protein scaffold.

Graphic abstract

中文翻译：

---

一种具有 DNA 切割活性的从头双核锌酶

摘要

金属水解酶在整个生物学中被广泛使用，通常用于催化大分子（如 DNA 和蛋白质）的降解。许多这些酶在其活性位点与锌一起起作用，并且这些酶的重要子集利用双核锌活性位点。已经开发了这些酶的模拟物，其中一些酶催化 DNA 的消化。然而，大多数利用锌的模拟物是小分子，而且大多数是单核的。在此，我们报告了 de novo 设计的Due Ferri 的DNA 切割活性含有双核锌活性位点的单链 (DFsc) 蛋白。这种双核锌-蛋白复合物能够以高达 50 ng/h 的速率消化质粒 DNA，并且这些裂解速率受锌结合位点附近氨基酸残基变化的影响。这些结果表明 DFsc 支架是一个很好的模型系统，可以进行仔​​细的结构 - 功能关系研究，以了解影响天然双核锌水解酶反应性的关键结构特征，因为它是蛋白质支架中双核模型系统的第一份报告.

图形摘要