論文

査読有り
2006年11月10日

Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltansferase) enzymes

Journal of Biological Chemistry
  • Kazunori Watanabe
  • ,
  • Osamu Nureki
  • ,
  • Shuya Fukai
  • ,
  • Yaeta Endo
  • ,
  • Hiroyuki Hori

281
45
開始ページ
34630
終了ページ
34639
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1074/jbc.M606141200

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2′-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

リンク情報
DOI
https://doi.org/10.1074/jbc.M606141200
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/16963456

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