Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family

Kazunori Watanabe, Osamu Nureki, Shuya Fukai, Ryohei Ishii, Hironori Okamoto, Shigeyuki Yokoyama, Yaeta Endo, Hiroyuki Hori

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54 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)10368-10377
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number11
DOIs
Publication statusPublished - Mar 18 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Watanabe, K., Nureki, O., Fukai, S., Ishii, R., Okamoto, H., Yokoyama, S., Endo, Y., & Hori, H. (2005). Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family. Journal of Biological Chemistry, 280(11), 10368-10377. https://doi.org/10.1074/jbc.M411209200