Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family
Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family
复制标题
DOI：
10.1074/jbc.m411209200
复制
发表时间：
2005-03-18
影响因子：
4.8
通讯作者：
Hori, H
中科院分区：
文献类型：
--
作者：
Watanabe, K;Nureki, O;Hori, H
转移RNA （Gm18）甲基转移酶（TrmH (SpoU)）催化一个甲基从s -腺苷- l-蛋氨酸（AdoMet）转移到tRNA中鸟苷18的2'- oh。该酶是RNA甲基转移酶SpoU家族的一员。最近的计算研究表明，SpoU成员之间有三个氨基酸序列基序是保守的。最近，我们确定了TrmH的apo-和AdoMet结合形式的晶体结构（Nureki， O., Watanabe， K., Fukai， S., Ishii， R., Endo， Y., Hori， H.，和Yokoyama， S. (2004) Structure 12, 593-602）。进一步明确了AdoMet的结合位点，提出了AdoMet的催化机理。由于基序中保守氨基酸残基的功能尚不清楚，我们制备了17个TrmH突变体，并进行了各种生化研究，包括测定AdoMet和tRNA的动力学参数，s -腺苷- l-同型半胱氨酸亲和层析，凝胶迁移率转移测定，CD光谱和分析凝胶过滤。我们的研究结果表明Asn（35）、Arg（41）、Glu（124）和Asn（152）参与tRNA的结合，Asn（35）残基参与s -腺苷- l-同型半胱氨酸的释放。TrmH的几个残基对酶的稳定性很重要。综上所述，我们的生化研究强化了之前提出的催化机制。我们还讨论了甲基转移酶的SPOUT超家族中的氨基酸取代。
Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl- L-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apo- and AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl- L-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl- L-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.