Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline
Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline
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DOI：
10.1021/bi0602314
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发表时间：
2006-06-20
期刊：
影响因子：
2.9
通讯作者：
Hou, Ya-Ming
中科院分区：
文献类型：
--
作者：
Christian, Thomas;Evilia, Caryn;Hou, Ya-Ming
tRNA（m1G37）甲基转移酶催化s -腺苷蛋氨酸（AdoMet）的一个甲基转移到G37的N1位置，即反密码子序列的3'，其修饰对于保持阅读框保真度很重要。虽然该酶在细菌中高度保守，由trmD基因编码，但最近的研究表明，该酶在古细菌和真核生物中由trm5基因编码，在序列和结构上都与trmD无关。为了进一步验证这一预测，我们试图确定催化所需的第二类tRNA（m1G37）甲基转移酶中的残基。这样的残馀应该提供对这两个类的不同结构起源的机械见解。以古菌Methano-caldococcus jannaschii（以前的MJ0883）的Trm5酶为例，我们创建了突变体来测试许多保守残基对AdoMet和tRNA的催化潜力和底物结合能力。我们发现267位脯氨酸（P267）是催化的关键残基，因为该残基的取代在稳态动力学分析中严重降低了甲基化反应的k(cat)，在单次翻转动力学分析中严重降低了k（chem）。而取代P267对K-m的影响较小，对两种底物的K-d的影响较小。由于P267没有可以直接参与甲基转移化学的功能侧链，我们认为它的催化作用是稳定酶和底物的构象，使酶活性位点的反应基团正确排列。序列分析表明，P267嵌入在Trm5家族中保守的肽基序中，但在TrmD家族中不存在，这支持了这两个家族是不相关蛋白质结构的后代的观点。
The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methano-caldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the k(cat) of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the K-m and little effect on the K-d of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.