近年来,形成具有调节的酶活性的丝状组件的酶已获得越来越多的关注。SgrAI是序列特异性II型限制性内切核酸酶,其形成具有加速的DNA切割活性和扩展的DNA序列特异性的聚合丝。先前的研究已经提出了一种机制模型,该模型将伴随SgrAI成丝的结构变化与其加速的DNA切割活性联系起来。在这个模型中,特定于丝状SgrAI的构象变化使细丝内不同酶拷贝之间的接触最大化,并在每个亚基中产生第二个二价阳离子结合位点,这反过来促进了DNA切割反应。然而,我们对原子催化机制的理解是不完整的。在这里,我们提出了使用低温电子显微镜(cryo-EM)解决的丝状SgrAI的两种新结构。第一种结构,解析为3.3µ,是丝状的SgrAI,含有活性位点突变,旨在阻止DNA切割反应,这揭示了DNA切割之前的酶构型。第二种结构,解析为3.1,是含有切割底物DNA的WT丝状SgrAI,这揭示了酶裂解反应结束时的酶构型。两种结构都含有裂解位点处的磷酸部分和生物学相关的二价阳离子辅因子Mg2+,并定义了Mg2+阳离子在酶催化过程中如何重新配置。数据支持激活机制的模型,该模型涉及在SgrAI活性位点中第二个Mg2结合,这是成丝诱导的构象变化的直接结果。
Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.