TY - JOUR
T1 - Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family
AU - Watanabe, Kazunori
AU - Nureki, Osamu
AU - Fukai, Shuya
AU - Ishii, Ryohei
AU - Okamoto, Hironori
AU - Yokoyama, Shigeyuki
AU - Endo, Yaeta
AU - Hori, Hiroyuki
PY - 2005/3/18
Y1 - 2005/3/18
N2 - 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, Arg41, Glu124, and Asn152 are involved in binding tRNA and that the Asn35 residue is involved in the release off 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.
AB - 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, Arg41, Glu124, and Asn152 are involved in binding tRNA and that the Asn35 residue is involved in the release off 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.
UR - http://www.scopus.com/inward/record.url?scp=15444370839&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=15444370839&partnerID=8YFLogxK
U2 - 10.1074/jbc.M411209200
DO - 10.1074/jbc.M411209200
M3 - Article
C2 - 15637073
AN - SCOPUS:15444370839
VL - 280
SP - 10368
EP - 10377
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 11
ER -