Christian Thomas, Evilia Caryn, Hou Ya-Ming
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 2006 Jun 20;45(24):7463-73. doi: 10.1021/bi0602314.
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 Methanocaldococcus 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 kcat 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 Km and little effect on the Kd 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.
tRNA（m1G37）甲基转移酶催化将一个甲基从S-腺苷甲硫氨酸（AdoMet）转移至G37的N1位，G37位于反密码子序列的3'端，其修饰对于维持读码框保真度很重要。虽然细菌中的该酶高度保守，由trmD基因编码，但最近的研究表明，古菌和真核生物中该酶的对应物由trm5基因编码，在序列和结构上均与trmD无关。为了进一步验证这一预测，我们试图鉴定第二类tRNA（m1G37）甲基转移酶中催化所需的残基。这些残基应能为这两类酶不同的结构起源提供机制上的见解。以嗜热栖热菌的Trm5酶（先前的MJ0883）为例，我们构建了突变体，以测试许多保守残基在AdoMet和tRNA方面的催化潜力和底物结合能力。我们发现，第267位的脯氨酸（P267）是催化的关键残基，因为在稳态动力学分析中，该残基的取代会严重降低甲基化反应的kcat，在单周转动力学分析中会降低k(chem)。然而，P267的取代对Km的影响较小，对任何一种底物的Kd几乎没有影响。由于P267没有可直接参与甲基转移化学过程的功能性侧链，我们认为它在催化中的作用是稳定酶和底物的构象，以便活性位点的反应基团正确排列。序列分析表明，P267嵌入在Trm5家族中保守但TrmD家族中不存在的肽基序中，支持了这两个家族是不相关蛋白质结构后代的观点。
