Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases
Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases
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DOI：
10.1093/nar/gkh564
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发表时间：
2004-04-01
影响因子：
14.9
通讯作者：
Redman, KL
中科院分区：
文献类型：
--
作者：
Bujnicki, JM;Feder, M;Redman, KL
三种类型的甲基转移酶（MTases）在核酸中产生5-甲基嘧啶，在RNA中形成m(5)U，在RNA中形成m(5)C，在DNA中形成m(5)C。DNA:m(5)C酶已通过晶体学、生物物理、生化和计算方法进行了广泛的研究。另一方面，RNA:m(5)C MTases的序列-结构-功能关系仍然不清楚，三种类型的5-甲基嘧啶生成酶之间的潜在进化关系也是如此。酵母tRNA:m(5)C MTase Trm4p（也称为Ncl1p）的序列分析和同源性建模为鉴定催化残基和建立RNA:m(5)C MTase活性位点的结构模型提供了结构和进化平台。除了先前发现的对Trm4p活性至关重要的基序IV和基序VI中的保守Cys残基外，分析还鉴定了两个对Trm4p活性很重要的不变残基。新发现的残基包括基序I中的赖氨酸残基和基序IV中的Asp残基。在基序X中发现的一个保守的Gln被发现对MTase活性是必不可少的。Trm4p模型中必需残基的位置与RNA:m(5)C MTase同源物PH1374的x射线结构非常吻合。理论和实验分析表明，RNA:m(5)C MTases与RNA:m(5)U MTases或DNA:m(5)C MTases具有许多共同的特征，这提示了这三类5-甲基嘧啶MTases之间关系的初步系统发育模型。我们推断，RNA:m(5)C MTases是从RNA:m(5)U MTases进化而来的，通过在基序IV中获得一个额外的Cys残基，该残基后来才在DNA:m(5)C MTases中发挥亲核催化剂的作用，伴随着基序VI中原始Cys的丢失，基序IV向基序VI的保守羧酸转移以及序列排列。
Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m(5)U in RNA, m(5)C in RNA and m(5)C in DNA. The DNA:m(5)C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m(5)C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m(5)C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m(5)C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m(5)C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m(5)C MTases share a number of features with either RNA:m(5)U MTases or DNA:m(5)C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m(5)C MTases evolved from RNA:m(5)U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m(5)C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.