Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes.
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.
Watanabe
Kazunori
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan.
Nureki
Osamu
O
Fukai
Shuya
S
Endo
Yaeta
Y
Hori
Hiroyuki
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017124:Conserved Sequence; D024202:Electrophoretic Mobility Shift Assay; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D012436:S-Adenosylmethionine; D012343:RNA, Transfer; D001120:Arginine; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase; D008239:Lysine