Active site dynamics of the Hhal methyltransferase:: Insights from computer simulation
Active site dynamics of the Hhal methyltransferase:: Insights from computer simulation
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DOI：
10.1006/jmbi.1999.3120
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发表时间：
1999-10-15
影响因子：
5.6
通讯作者：
Bruice, TC
中科院分区：
文献类型：
--
作者：
Lau, EY;Bruice, TC
用分子动力学方法研究了DNA甲基化转移酶M.HhaI与DNA和ADOMet形成的三元络合物。甲基化包括将Cys81的巯基阴离子加到Cyt18的6位上，然后生成的碳负离子在C5上对ADOMet甲基进行亲核攻击。在模拟中发现，Cys81的巯基(SG)到Cyt18的C6(SG-C6)和ADOMet的甲基碳(CH3)到胞嘧啶的C5(CH3-C5)之间的距离取决于核苷酸的二面角chi(O4‘-C1’-N1-C2)。当目标胞嘧啶残基的Cytl8的X角较低(-80度)时，SG-C6和CH3-C-5的距离都减小，胞嘧啶环和ADOMet之间形成的角度与从头计算胞嘧啶甲基化过程中形成的过渡态结构的值很好地对应。在模拟中发现了两种可能的质子源，用于胞嘧啶残基形成共价中间体时N3的质子化。ADOMet的质子化胺基团可以通过水桥提供质子，或者Arg163也可以通过水桥提供N3的质子来源。该模拟提供了1-15个胞嘧啶如何从活性中心进入溶剂的洞察力。保守残基Asn304和Gln82稳定了M.HhaI活性中心内的水网络，这为1-15扩散到本体溶剂中提供了一条途径。最初距离较远的水分子能够扩散到酶的活性部位，并取代接近胞嘧啶C5的结晶性水分子的位置。这种水分子的运动表明，在M.HhaI的Gln82和Adobe Met之间存在一条通道，使水和质子都可以很容易地进入酶的活性部位。(C)1999年学术出版社。
A molecular dynamics study was performed on the DNA methyltransferase M.HhaI in a ternary complex with DNA and AdoMet in solution. Methylation involves addition of the Cys81 sulfhydryl anion to the 6-position of Cyt18, followed by a nucleophilic attack of the resultant carbanion at C5 on the AdoMet methyl group. It was found in this simulation that the distances between the sulfhydryl group (SG) of Cys81 to the C6 of Cyt18 (SG-C6) and methyl carbon (CH3) of AdoMet to the C5 of cytosine (CH3-C5) are dependent on the dihedral angle chi (O4'-C1'-N1-C2) of the nucleotide. When the chi angle of Cytl8 is low (-80 degrees) for the target cytosine residue reduces the distances for both SG-C6 and CH3-C-5, and the angles formed between the cytosine ring and AdoMet correspond well to values for the transition state structures formed during methylation of cytosine from ab initio calculations. Two possible proton sources for protonation of N3 of the cytosine residue upon formation of the covalent intermediate were found in the simulation. The protonated amine group of AdoMet could provide a proton via a water bridge, or Arg163 could also be the source of the proton for N3 via a water bridge. The simulation provides insights into how the 1-15 of cytosine could go from the active site into solvent. Conserved residues Asn304 and Gln82 stabilize a water network within the active site of M.HhaI which provides a route for 1-15 to diffuse into bulk solvent. An initially distant water molecule was able to diffuse into the active site of the enzyme and replace a position of a crystallographic water molecule in close proximity to the C5 of cytosine. The movement of this water molecule showed that a channel exists between Gln82 and the AdoMet in M.HhaI which allows both water and protons to easily gain access to the active site of the enzyme. (C) 1999 Academic Press.