Cheng X, Balendiran K, Schildkraut I, Anderson J E
W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, NY 11724.
EMBO J. 1994 Sep 1;13(17):3927-35. doi: 10.1002/j.1460-2075.1994.tb06708.x.
We have determined the structure of PvuII endonuclease complexed with cognate DNA by X-ray crystallography. The DNA substrate is bound with a single homodimeric protein, each subunit of which reveals three structural regions. The catalytic region strongly resembles structures of other restriction endonucleases, even though these regions have dissimilar primary sequences. Comparison of the active site with those of EcoRV and EcoRI endonucleases reveals a conserved triplet sequence close to the reactive phosphodiester group and a conserved acidic pair that may represent the ligands for the catalytic cofactor Mg2+. The DNA duplex is not significantly bent and maintains a B-DNA-like conformation. The subunit interface region of the homodimeric protein consists of a pseudo-three-helix bundle. Direct contacts between the protein and the base pairs of the PvuII recognition site occur exclusively in the major groove through two antiparallel beta strands from the sequence recognition region of the protein. Water-mediated contacts are made in the minor grooves to central bases of the site. If restriction enzymes do share a common ancestor, as has been proposed, their catalytic regions have been very strongly conserved, while their subunit interfaces and DNA sequence recognition regions have undergone remarkable structural variation.
我们通过X射线晶体学确定了与同源DNA复合的PvuII核酸内切酶的结构。DNA底物与单个同型二聚体蛋白结合，该蛋白的每个亚基都有三个结构区域。催化区域与其他限制性核酸内切酶的结构非常相似，尽管这些区域的一级序列不同。将活性位点与EcoRV和EcoRI核酸内切酶的活性位点进行比较，发现靠近反应性磷酸二酯基团处有一个保守的三联体序列，以及一对保守的酸性基团，它们可能是催化辅因子Mg2+的配体。DNA双链没有明显弯曲，保持着类似B-DNA的构象。同型二聚体蛋白的亚基界面区域由一个假三螺旋束组成。蛋白质与PvuII识别位点的碱基对之间的直接接触仅通过来自蛋白质序列识别区域的两条反平行β链在大沟中发生。在小沟中与位点的中央碱基形成水介导的接触。如果如所提出的那样，限制性酶确实共享一个共同的祖先，那么它们的催化区域一直非常保守，而它们的亚基界面和DNA序列识别区域则经历了显著的结构变化。
