Lagerbäck Pernilla, Carlson Karin
Department of Cell and Molecular Biology, Uppsala University, Box 596, Uppsala, Sweden.
J Bacteriol. 2008 Aug;190(16):5533-44. doi: 10.1128/JB.00094-08. Epub 2008 Jun 6.
Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.
噬菌体T4核酸内切酶II（EndoII）是一种缺乏羧基末端DNA结合结构域的GIY-YIG核酸内切酶，通过定点诱变来研究单个氨基酸在底物识别、结合和催化中的作用。EndoII的结构是以UvrC的结构为模型构建的。我们发现，假定催化表面上的残基（G49、R57、E118和N130）具有与I-TevI和UvrC中所描述的类似的催化作用；此外，还发现这些残基对于底物识别和结合很重要。保守的甘氨酸（G49）和精氨酸（R57）对于正常的序列识别至关重要。我们的结果与这些残基在形成DNA结合表面以及在活性位点暴露底物可切割键方面所起的作用一致。保守的天冬酰胺（N130）和相邻的脯氨酸（P127）可能有助于正确定位催化结构域。GIY-YIG核酸内切酶EndoII亚家族中的酶共享一个高度保守的中间区域（MR，残基72至93，可能是螺旋结构，可能替代I-TevI和UvrC中的异源螺旋）和一个保守性稍弱的N端区域（残基12至24）。这两个区域中的大多数保守残基似乎有助于结合强度，而不影响催化表面上底物结合的模式。EndoII的K76是归巢核酸内切酶保守的NUMOD3 DNA结合基序的一部分，发现它与MR重叠，影响序列识别和催化，表明其更直接地参与底物定位。因此，我们的数据表明GIY-YIG酶中保守的MR和残基在识别和结合底物方面发挥作用。
