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J. Biol. Chem., Vol. 281, Issue 45, 34630-34639, November 10, 2006

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Functional Categorization of the Conserved Basic Amino Acid Residues in TrmH (tRNA (Gm18) Methyltansferase) Enzymes^*

Kazunori Watanabe¹, Osamu Nureki, Shuya Fukai, Yaeta Endo^¶||, and Hiroyuki Hori^¶2

From the Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan, Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan, ^¶Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan, and ^||Cell-free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan

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 K_d and K_m 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.

Received for publication, June 27, 2006 , and in revised form, September 6, 2006.

^* This work was partly supported by Grant-in-aid 18048032 for Science Research on Priority Areas (to H. H.), Grant-in-aid 17613003 for Science Research from the Ministry of Education, Science, Sports, and Culture of Japan (to H. H.), Sumitomo Foundation Grant for Basic Science Research Projects 050137 (to H. H.), and a Grant from the Circle for the Promotion of Science and Engineering (to K. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Functional Nucleic Acids Research Group, Institute for Biological Resources and Functions, National Institute for Advanced Industrial Science and Technology, 1-1-1, Higashi, Tsukuba, Ibaragi, 305-8566, Japan.

² To whom correspondence should be addressed. Tel.: 81-89-927-8548; Fax: 81-89-927-9941; E-mail: hori{at}eng.ehime-u.ac.jp.

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