Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase
Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase
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DOI：
10.1016/j.jmb.2006.01.035
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发表时间：
2006-03-31
影响因子：
5.6
通讯作者：
Jeltsch, A
中科院分区：
文献类型：
--
作者：
Gowher, H;Loutchanwoot, P;Jeltsch, A
根据氨基酸序列比对和相关酶的结构数据，我们对小鼠DNMT3A DNA-(胞嘧啶C5)-甲基转移酶催化区域的14个氨基酸残基进行了突变分析。目标残基位于胞嘧啶-C5甲基转移酶特有的10个保守氨基酸序列基序中，并位于该酶的DNA识别结构域(TRD)。对突变蛋白进行纯化，并检测其催化性能及其与DNA和ADOMet的结合能力。我们制备了DNMT3A的结构模型。来解释我们的结果。我们证明了Phe50(基序I)和Glu74(基序II)对于Adobe Met的结合和催化是重要的。在与S-腺苷-L-蛋氨酸(ADOMet)饱和的条件下，D96A(Motif III)的结合活性降低，但活性增加，表明Asp96不是催化所必需的。R130A(在基序IV之后)、R241A和R246A(在TRD中)、R292A和R297A(都位于基序X的前面)显示DNA结合减少。R130A的催化活性显著降低，侧翼序列偏好完全改变，表明Arg130在DNMT3A的DNA相互作用中起着重要作用。R292a还表现出活性降低和侧翼序列偏好的变化，表明在远离CG靶点的DNA接触中可能发挥了作用。N167A(基序VI)和R202A(基序VIII)具有正常的ADOMet和DNA结合，但催化活性降低。虽然Asn167可能有助于基序VI的残基定位，但根据结构数据，Arg202在胞嘧啶-C5甲基转移酶的催化中发挥作用。R295A变异体催化失活，很可能是因为蛋白质的铰链亚结构域不稳定。(C)2006爱思唯尔有限公司。保留所有权利。
On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a. to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein. (c) 2006 Elsevier Ltd. All rights reserved.