Enzymatic C5-cytosine methylation of DNA: Mechanistic implications of new crystal structures for HhaI methyltransferase-DNA-AdoHcy complexes
Enzymatic C5-cytosine methylation of DNA: Mechanistic implications of new crystal structures for HhaI methyltransferase-DNA-AdoHcy complexes
复制标题
DOI：
10.1006/jmbi.1996.0489
复制
发表时间：
1996-09-06
影响因子：
5.6
通讯作者：
Cheng, XD
中科院分区：
文献类型：
--
作者：
OGara, M;Klimasauskas, S;Cheng, XD
HhaI甲基转移酶与同源未甲基化或甲基化DNA以及S-腺苷-L-高半胱氨酸复合的精细晶体结构，沿着先前解决的二元和共价三元结构，提供了整个反应周期各个阶段活性位点的详细图像。这张图片支持并扩展了C5-胞嘧啶甲基化的拟议机制，该机制可能是整个C5-胞嘧啶甲基转移酶家族的通用机制。两个新的配合物的结构已被细化到晶体学的R-因子分别为0.189和0.178，在2.7埃分辨率。我们观察到未甲基化的2 '-脱氧胞苷和5-甲基-2'-脱氧胞苷都从DNA螺旋中翻转出来，并进入酶的活性位点。Cys 81的催化硫原子与C6强烈相互作用。翻转的5-甲基-2 '-脱氧胞苷的C5甲基基团被弯曲成类似于50度离开胞嘧啶环的平面并朝向S-腺苷-L-高半胱氨酸的硫原子。这一不寻常的位置可能是由于C5和C6的部分sp（3）特征以及保守氨基酸残基Pro 80和Cys 81的空间效应。两个水分子通过两个保守的氨基酸残基（Gln 82和Asn 304）和翻转核苷酸3'的磷酸基团的磷酰基氧原子保持在翻转胞嘧啶的疏水边缘（C5和C6）附近，并且其中一个可以作为从C5消除质子的通用碱基。甲基化反应期间胞嘧啶N3的质子化可能涉及Glu 119，Glu 119本身可能通过Glu 119的末端羧基与S-腺苷-L-甲硫氨酸的甲硫氨酸部分的氨基之间的CTATER介导的相互作用而质子化。因此，辅因子在反应中起着两个关键作用。(C)1996年学术出版社
The refined crystal structures of HhaI methyltransferase complexed with cognate unmethylated or methylated DNA together with S-adenosyl-L-homocysteine, along with the previously-solved binary and covalent ternary structures, offer a detailed picture of the active site at individual stages throughout the reaction cycle. This picture supports and extends a proposed mechanism for C5-cytosine methylation that may be general for the whole family of C5-cytosine methyltransferases. The structures of the two new complexes have been refined to crystallographic R-factors of 0.189 and 0.178, respectively, at 2.7 Angstrom resolution. We observe that both unmethylated 2'-deoxycytidine and 5-methyl-2'-deoxycytidine flip out of the DNA helix and fit into the active site of the enzyme. The catalytic sulfur atom of Cys81 interacts strongly with C6. The C5 methyl group of the flipped 5-methyl-2'-deoxycytidine is bent similar to 50 degrees out of the plane of the cytosine ring and towards the sulfur atom of S-adenosyl-L-homocysteine. This unusual position is probably due to partial sp(3) character at C5 and C6 and to steric effects of the conserved amino acid residues Pro80 and Cys81. Two water molecules are held near the hydrophobic edge (C5 and C6) of the flipped cytosine by two conserved amino acid residues (Gln82 and Asn304) and the phosphoryl oxygen atom of the phosphate group 3' to the flipped nucleotide, and one of them may serve as the general base for eliminating the proton from C5. Protonation of the cytosine N3 during the methylation reaction may involve Glu119, which itself might be protonated via a ct ater-mediated interaction between the terminal carboxyl group of Glu119 and the amino group of the methionine moiety of S-adenosyl-L-methionine. The cofactor thus plays two key roles in the reaction. (C) 1996 Academic Press Limited