Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.
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 apoand 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.
Watanabe
Kazunori
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan.
Nureki
Osamu
O
Fukai
Shuya
S
Ishii
Ryohei
R
Okamoto
Hironori
H
Yokoyama
Shigeyuki
S
Endo
Yaeta
Y
Hori
Hiroyuki
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology; D013997:Time Factors; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000502:physiology
D001216:Asparagine; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase; D003545:Cysteine