Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction-modification (R-M) systems and several of them are strain specific and phase variable. HpyAII is a novel Type IIs phase variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first His residue of the HNH motif to Ala does not abolish the enzymatic activity, but instead causes loss of fidelity compared to wild type HpyAII. Previous studies have shown that mutation of the first His residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. This study provides further insights into the evolution of restriction enzymes.

中文翻译：

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HpyAII内切核酸酶的混杂DNA切割受HNH催化残基调控。

幽门螺杆菌是致癌细菌，占所有人类胃癌的5.5％。幽门螺杆菌编码用于异常大量的限制修饰（RM）系统，其中一些是特定于应变和相位可变的。HpyAII是一种新型的IIs型相变限制性核酸内切酶，存在于幽门螺杆菌26695株中。我们显示，HpyAII较之于一个位点的底物，其最大的裂解活性更偏爱两个位点的底物。HpyAII对金属离子的要求不那么严格，并且与Nig +相比，对Mg2 +的裂解活性更高。对HpyAII的HNH基序的假定残基进行突变分析，证实该蛋白具有一个可用于DNA切割的活性HNH位点。但是，将HNH基序的第一个His残基突变为Ala不会消除酶促活性，但与野生型HpyAII相比，却会导致保真度下降。先前的研究表明，所有其他已知的包含HNH基序基序的酶的HNH基序的第一个His残基的突变完全消除了酶促活性。我们发现，在HpyAII的情况下，活性位点残基的突变会导致核酸内切酶保真度的损失。这项研究为限制酶的进化提供了进一步的见解。