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• W2018073387 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 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 Asn35, Arg41, Glu124, and Asn152 are involved in binding tRNA and that the Asn35 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. 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 Asn35, Arg41, Glu124, and Asn152 are involved in binding tRNA and that the Asn35 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. Transfer RNA (Gm18) methyltransferase (TrmH (classical name, SpoU); tRNA (guanosine-2′-)-methyltransferase, EC 2.1.1.34) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; AdoHcy, S-adenosyl-l-homocysteine. to the 2′-OH of the ribose of guanosine at position 18 (G18) in the D-loop of tRNA (1Kumagai I. Watanabe K. Oshima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar, 2Kumagai I. Watanabe K. Oshima T. J. Biol. Chem. 1982; 257: 7388-7395Abstract Full Text PDF PubMed Google Scholar, 3Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. (Tokyo). 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 4Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. (Tokyo). 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar, 5Hori H. Yamazaki N. Matsumoto T. Watanabe Y. Ueda T. Nishikawa K. Kumagai I. Watanabe K. J. Biol. Chem. 1998; 273: 25721-25727Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar, 7Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 3969-3973Crossref PubMed Scopus (81) Google Scholar, 9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar, 10Cavaille J. Chetouani F. Bachellerie J.-P. RNA. 1999; 5: 66-81Crossref PubMed Scopus (80) Google Scholar). G18 is a highly conserved residue in the D-loop of tRNA (11McCloskey J.A. Crain P.F. Nucleic Acids Res. 1998; 26: 196-197Crossref PubMed Scopus (70) Google Scholar, 12Rozenski J. McCloskey J.A. Crain P.F. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (350) Google Scholar) and is responsible for the formation of the L-shaped three-dimensional structure by D-loop/T-loop interaction through the G18-Ψ55 tertiary base pair (13Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F.C. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (803) Google Scholar, 14Kim S.H. Sussman J.L. Suddath F.L. Quigley G.J. McPherson A. Wang A.H.J. Seeman N.C. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4970-4974Crossref PubMed Scopus (233) Google Scholar). Because the 2′-O-methylation of ribose at position 34 stabilizes the C3′-endo form (15Kawai G. Yamamoto Y. Kamimura T. Masegi T. Sekine M. Hata T. Iimori T. Watanabe T. Miyazawa T. Yokoyama S. Biochemistry. 1992; 31: 1040-1046Crossref PubMed Scopus (173) Google Scholar), the 2′-O-methyl-G18 (Gm18) modification may confer conformational rigidity on the local structure of RNA. Absence of the Gm18 modification in E. coli tRNA has no effect on the activity of the supF amber suppressor tRNA or on the growth rate of cells (8Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 3969-3973Crossref PubMed Scopus (81) Google Scholar); however, it has been recently reported that the growth rate in an E. coli mutant lacking both Gm18 and Ψ55 is decreased and that the frequency of the frameshift errors in the mutant strain is increased (16Urbonavicius J. Durand J.M. Björk G.R. J. Bacteriol. 2002; 184: 5348-5357Crossref PubMed Scopus (48) Google Scholar). Thus, the Gm18 modification probably works in conjunction with the other modified nucleoside(s). The Gm18 modification in tRNA is distributed in eubacteria and eukaryotes and is also found in plant organella (11McCloskey J.A. Crain P.F. Nucleic Acids Res. 1998; 26: 196-197Crossref PubMed Scopus (70) Google Scholar, 12Rozenski J. McCloskey J.A. Crain P.F. Nucleic Acids Res. 1999; 27: 196-197Crossref PubMed Scopus (350) Google Scholar). The genes responsible for the Gm18 modification have been identified as trmH (spoU) in Escherichia coli (8Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 3969-3973Crossref PubMed Scopus (81) Google Scholar, 9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar), Thermus thermophilus (6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar), and Aquifex aeolicus (7Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and as trm3 in Saccharomyces cerevisiae (10Cavaille J. Chetouani F. Bachellerie J.-P. RNA. 1999; 5: 66-81Crossref PubMed Scopus (80) Google Scholar). Recent analyses based on the amino acid sequence alignments have shown that several RNA ribose 2′-O-methyltransferases share the common sequence motifs (termed motifs 1, 2, and 3) and thus have been classified as SpoU family methyltransferases (9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar). All of the enzymes characterized in the SpoU family, including T. thermophilus TrmH, are involved in the ribose methylation of tRNA (1Kumagai I. Watanabe K. Oshima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar, 2Kumagai I. Watanabe K. Oshima T. J. Biol. Chem. 1982; 257: 7388-7395Abstract Full Text PDF PubMed Google Scholar, 3Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. (Tokyo). 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 4Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. (Tokyo). 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar, 5Hori H. Yamazaki N. Matsumoto T. Watanabe Y. Ueda T. Nishikawa K. Kumagai I. Watanabe K. J. Biol. Chem. 1998; 273: 25721-25727Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar, 7Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8Persson B.C. Jager G. Gustafsson C. Nucleic Acids Res. 1997; 25: 3969-3973Crossref PubMed Scopus (81) Google Scholar, 9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar, 10Cavaille J. Chetouani F. Bachellerie J.-P. RNA. 1999; 5: 66-81Crossref PubMed Scopus (80) Google Scholar) or rRNA (17Sirum-Connolly K. Mason T.L. Science. 1993; 262: 1886-1889Crossref PubMed Scopus (100) Google Scholar, 18Thompson J. Schmidt F. Cundliffe E. J. Biol. Chem. 1982; 257: 7915-7917Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). T. thermophilus TrmH has many merits as a model of SpoU family for structural and functional studies of the family, because this enzyme is a small, stable, and well characterized member of the family (1Kumagai I. Watanabe K. Oshima T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1922-1926Crossref PubMed Scopus (36) Google Scholar, 2Kumagai I. Watanabe K. Oshima T. J. Biol. Chem. 1982; 257: 7388-7395Abstract Full Text PDF PubMed Google Scholar, 3Hori H. Saneyoshi M. Kumagai I. Miura K. Watanabe K. J. Biochem. (Tokyo). 1989; 106: 798-802Crossref PubMed Scopus (23) Google Scholar, 4Matsumoto T. Nishikawa K. Hori H. Ohta T. Miura K. Watanabe K. J. Biochem. (Tokyo). 1990; 107: 331-338Crossref PubMed Scopus (25) Google Scholar, 5Hori H. Yamazaki N. Matsumoto T. Watanabe Y. Ueda T. Nishikawa K. Kumagai I. Watanabe K. J. Biol. Chem. 1998; 273: 25721-25727Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar). In a recent study, we determined the crystal structures of the AdoMet-binding and the apo forms of T. thermophilus TrmH (19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We also carried out an alanine substitution experiment to clarify the residues involved in the AdoMet-binding site (19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Furthermore, we have proposed a novel catalytic mechanism for the enzyme (19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) (see Fig. 6).Fig. 6Schematic drawing of the hypothetical catalytic mechanism of TrmH. On the basis of the crystal structure of the AdoMet, sulfate, and TrmH complex, we proposed the hypothetical catalytic mechanism shown here (19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The detailed mechanism has been described previously (19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The AdoMet binding subunit (monomer 1) and tRNA binding subunit (monomer 2) are indicated in black and gray, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The crystal structure revealed that the C-terminal region of the enzyme forms a deep trefoil knot structure, which is produced by threading the polypeptide chain through an untwisted loop (20Nureki O. Shirouzu M. Hashimoto K. Ishitani R. Terada T. Tamakoshi M. Oshima T. Chijimatsu M. Takio K. Vassylyev D.G. Shibata T. Inoue Y. Kuramitsu S. Yokoyama S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1129-1137Crossref PubMed Scopus (125) Google Scholar). Our results are consistent with recent reports that several SpoU proteins (T. thermophilus RrmA (20Nureki O. Shirouzu M. Hashimoto K. Ishitani R. Terada T. Tamakoshi M. Oshima T. Chijimatsu M. Takio K. Vassylyev D.G. Shibata T. Inoue Y. Kuramitsu S. Yokoyama S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1129-1137Crossref PubMed Scopus (125) Google Scholar), E. coli RmlB (21Michel G. Sauve V. Larocque R. Li Y. Matte A. Cygler M. Structure. 2002; 10: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), Methanobacterium thermoautotrophicum MT0001 (22Zarembinski T.I. Kim Y. Peterson K. Christendat D. Dharamsi A. Arrowsmith C.H. Edwards A.M. Joachimiak A. Proteins. 2003; 50: 177-183Crossref PubMed Scopus (39) Google Scholar), and Haemophilus influenzae YibK (23Lim K. Zhang H. Tempczyk A. Krajewski W. Bonander N. Toedt J. Howard A. Eisenstein E. Herzberg O. Proteins. 2003; 51: 56-67Crossref PubMed Scopus (87) Google Scholar)) have a deep trefoil knot in their C-terminal regions. During the course of the present work, it has been reported that the TrmD (tRNA (m1G37) methyltransferase (24Byström A.S. Björk G.R. Mol. Gen. Genet. 1982; 188: 440-446Crossref PubMed Scopus (56) Google Scholar, 25Byström A.S. Hjaimarsson K.J. Wikstrom P.M. Björk G.R. EMBO J. 1983; 2: 899-905Crossref PubMed Scopus (71) Google Scholar, 26Hjalmarsson K.J. Byström A.S. Björk G.R. J. Biol. Chem. 1983; 258: 1343-1351Abstract Full Text PDF PubMed Google Scholar, 27Holmes W.M. Andraos-Selim C. Roberts I. Wahab S.Z. J. Biol. Chem. 1992; 267: 13440-13445Abstract Full Text PDF PubMed Google Scholar, 28Redlak M. Andraos-Selim C. Giege R. Florentz C. Holmes W.M. Biochemistry. 1997; 36: 8699-8709Crossref PubMed Scopus (43) Google Scholar)) proteins in H. influenzae (29Ahn H.J. Kim H.W. Yoon H.J. Lee B. Suh S.S. Yang J.K. EMBO J. 2003; 22: 2593-2603Crossref PubMed Scopus (119) Google Scholar) and E. coli (30Elkins P.A. Watts J.M. Zalacain M. van Thiel A. Vitazka P.R. Redlak M. Andraos-Selim C. Rastinejad F. Holmes W.M. J. Mol. Biol. 2003; 333: 931-949Crossref PubMed Scopus (106) Google Scholar) also have a deep trefoil knot in their C-terminal region. The SpoU and TrmD families were previously considered to be unrelated to each other; however, recent computational analysis has suggested that they might share a common evolutionary origin and form a single “SPOUT” (SpoU-TrmD) superfamily (31Anantharaman V. Koonin E.V. Aravind L. J. Mol. Microbiol. Biotechnol. 2002; 4: 71-75PubMed Google Scholar) (Fig. 1). Taking the above information together, the trefoil knot structure is probably conserved in the catalytic domains of most SPOUT methyltransferases. This raises, however, an important question. The roles of the conserved amino acid residues in the motifs that are shared across the various SpoU members remain unclear. In this report, we demonstrate that the residues conserved in the motifs are involved in the catalytic mechanism of the enzyme, such as the binding of both substrates (AdoMet and tRNA) and the release of the products, and also contribute to the stability of the protein structure. Furthermore, we discuss amino acid substitutions in general among the SPOUT superfamily of methyltransferases. Materials—[methyl-14C]AdoMet (1.95 GBq/mmol) and [methyl-3H]AdoMet (2.47 TBq/mmol) were purchased from ICN; cold AdoMet and S-adenosyl-l-homocysteine (AdoHcy) were from Sigma; and DE52 was from Whatman. Sephacryl S-100 high resolution came from Amersham Biosciences. The AdoHcy affinity column was synthesized from a HiTrap NHS-activated HP column (Amersham Biosciences) according to the manufacturer's manual. CM-Toyopearl 650M was from Tosoh, Japan. DNA oligomers were bought from Invitrogen, and T7 RNA polymerase was from Toyobo, Japan. All other chemical reagents were of analytical grade. Site-directed Mutagenesis and Purification of the Proteins—The gene encoding tRNA (Gm18) methyltransferase from T. thermophilus HB8 was cloned into the E. coli pET30a expression vector (6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar). We carried out the site-directed mutagenesis by using a QuikChange mutagenesis kit (Stratagene). The sequences of the plasmids were analyzed with ABI PRISM 310 DNA sequencers. The wild-type enzyme was purified as reported previously (6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar). Twelve mutant proteins (N35A, N35Q, S37G, R41K, R41M, E124Q, E124D, P143Q, S150C, S150T, N152D, and N152E) were purified by the same method. Because the AdoHcy affinity column did not efficiently purify five mutant proteins (N35D, R41A, E124A, S150A, and N152A), we purified these proteins by two-step ion exchange chromatography. In brief, fractions of the DE52 column chromatography step in the standard purification procedure were combined, dialyzed against buffer A (50 mm HEPES-NaOH (pH 6.8), 5 mm MgCl2, and 6 mm 2-mercaptoethanol), and separated by CM-Toyopearl 650M column chromatography (liner gradient; 100–250 mm KCl in the buffer A). The mutant protein fractions were assessed by 15% SDS-polyacrylamide gel electrophoresis. In cases where the purity of the protein was not satisfactory, we repeated the CM-Toyopearl 650M column chromatography step after dialysis against the buffer A. The fractions were combined, dialyzed against buffer B (50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 6 mm mercaptoethanol, and 50 mm KCl), and concentrated with Centriprep YM-10 centrifugal filter devices (Millipore Corp.). Glycerol was added to the purified proteins to a final concentration of 50%, and the samples were stored at –30 °C. The quantity of protein was measured with a Bio-Rad protein assay kit using bovine serum albumin as the standard. Measurements of Enzymatic Activity—A standard assay for enzyme activity was carried out in which incorporation of the methyl group from [methyl-14C]AdoMet into yeast tRNAPhe transcript was measured as follows: 300 ng of protein, 7 μm transcript, and 38 μm [methyl-14C]AdoMet in 30 μl of buffer B were incubated for 5 min at 50 °C, and then an aliquot (20 μl) was assessed by the conventional filter assay. The yeast tRNAPhe transcript was prepared as reported previously (7Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The apparent kinetic parameters, Km and Vmax, were determined by a Lineweaver-Burk plot of the methyl transfer reaction with [methyl-3H]AdoMet. To determine the kinetic parameters for AdoMet, the concentrations of the mutant proteins and yeast tRNAPhe transcript were fixed at 0.23 and 8.5 μm, respectively. However, the range of the AdoMet concentrations and the incubation times were varied according to the methyl transfer activity of the protein. When the mutant protein had relatively high activity, [methyl-3H]AdoMet concentrations of 0, 1, 5, 10, 15, 20, and 30 μm were used. When the mutant proteins had relatively low activity, we measured the initial velocities under modified conditions as follows. The AdoMet concentrations were adjusted to 0, 0.25, 0.5, 0.75, 1.0, and 2.0 mm by the addition of cold AdoMet. In the latter case, the incubation period was prolonged to 60 min. Kinetic parameters for tRNA were determined as previously reported (7Hori H. Kubota S. Watanabe K. Kim J.M. Ogasawara T. Sawasaki T. Endo Y. J. Biol. Chem. 2003; 278: 25081-25090Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Analytical Ado-Hcy Affinity Column Chromatography—The affinity of the purified proteins for AdoHcy was qualitatively analyzed by the Ado-Hcy column chromatography as described in our previous report (6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google Scholar, 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Gel Mobility Shift Assay—Purified protein (final concentrations, 0, 1.1, 2.3, 3.4, 4.5, 5.7, 6.8, 8.0, 9.1, 10.0, and 11 μm) and 5 μm yeast tRNAPhe transcript were incubated in 20 μl of buffer C (25 mm Trisacetic acid (pH 7.0) and 5 mm Mg(OAc)2) at 37 °C for 20 min. A 6% polyacrylamide gel (width, 90 mm; length, 90 mm; thickness, 1 mm) was prepared with 1× buffer C, and 4 μl of loading solution (0.25% bromphenol blue and 30% glycerol) was added to each of the samples, which were then loaded onto the gel immediately. Electrophoresis was carried out at room temperature under a constant voltage (100 V) for 1 h. The gel was stained with Coomassie Brilliant Blue to detect protein and then with methylene blue to detect RNA. The quantities of RNA and protein were measured with a Fuji Photo Film BAS2000 imaging analyzer. Measurement of CD Spectra—CD spectra were measured with a JASCO spectropolarimeter J-820 equipped with a JASCO PTC-423L thermocontroller. Cuvettes with a 1-mm path length were used. Each sample (250 μg/ml) in buffer D (50 mm Tris-HCl (pH 7.6), 5 mm MgCl2, and 50 mm KCl) was preincubated at 25, 50, or 75 °C for 20 min, and then the spectrum was recorded from 300 to 200 nm at each temperature. The scan speed was 50 nm/min. The spectra shown in this paper are the average of five scans. The content of α-helix (percentage) was calculated according to Ref. 32Clark D.J. Hill C. Martin S.R. Thomas J.O. EMBO J. 1988; 7: 69-75Crossref PubMed Scopus (137) Google Scholar. Analytical Gel Filtration—Analytical gel filtration was performed with an ÄKTAprime chromatography system (Amersham Biosciences) equipped with a Superdex 75 column (10/30; column volume, 23.6 ml). The column was first equilibrated with buffer B containing 200 mm KCl, and the sample (30–50 μg) was then injected. The flow rate was 0.5 ml/min. The elution profiles were monitored by the absorption of UV at 280 nm. The enzyme-tRNA complex was analyzed as follows. The enzyme (400 pmol) and tRNA (80–800 pmol) were incubated at 4 °C for 5 min in 100 μl of the buffer B and then injected onto the column. The elution profiles were monitored by the absorption of UV at 260 and 280 nm. Selection of Target Sites for the Site-directed Mutagenesis— Fig. 1 schematically shows the multiple amino acid sequence alignment of the eubacterial SPOUT proteins that have been identified functionally and/or structurally. To generate the alignment, we basically followed the publications of Gustafson et al. (9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar) and Anantharaman et al. (31Anantharaman V. Koonin E.V. Aravind L. J. Mol. Microbiol. Biotechnol. 2002; 4: 71-75PubMed Google Scholar). Conserved amino acid sequences resembling motifs 1, 2, and 3 are also found in members of the TrmD family, as reported by Anantharaman (31Anantharaman V. Koonin E.V. Aravind L. J. Mol. Microbiol. Biotechnol. 2002; 4: 71-75PubMed Google Scholar) (Fig. 1), although these motifs were originally identified in SpoU family members (9Gustafsson C. Reid R. Greene P.J. Santi D.V. Nucleic Acids Res. 1996; 24: 3756-3762Crossref PubMed Scopus (120) Google Scholar). In the present work, we focused mainly on the amino acid residues conserved in these motifs. We therefore selected amino acid residues on this basis as the target sites for mutation; these residues are indicated in red in Fig. 1. Although the residues selected are separated in the primary amino acid sequence, they constitute the catalytic pocket in the three-dimensional structure (see Fig. 8). We prepared 17 mutants in this work (Table I) and expressed the proteins by the E. coli pET 30a expression system. All proteins were purified to homogeneity, as assessed by 15% SDS-polyacrylamide gel electrophoresis as described under “Experimental Procedures” (data not shown; see Figs. 2 and 7).Table IKinetic parameters for AdoMet and yeast tRNAPhe transcriptVariant nameAdoMettRNA transcriptReference/SourceKmVmaxRelative Vmax/KmKmVmaxμmμmol/mg·hnmμmol/mg·hWild type101011003Ref. 6Hori H. Suzuki T. Sugawara K. Inoue Y. Shibata T. Kuramitsu S. Yokoyama S. Oshima T. Watanabe K. Genes Cells. 2002; 7: 259-272Crossref PubMed Scopus (50) Google ScholaraKinetic parameters for AdoMet were previously reported in the reference.N35A830.510.006120000.54This workN35Q21000.0370.000018—b—, kinetic parameters for the transcript could not be calculated, because the affinity to AdoMet was very poor.—This workN35D6700.0160.000024——This workS37G51110.2231011This workR41A46000.0620.000013——Ref. 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google ScholaraKinetic parameters for AdoMet were previously reported in the reference.R41K3000.410.0041——Ref. 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google ScholarR41M9200.0760.000083——This workE124A28,0000.460.000016——Ref. 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google ScholarE124QNDcND, methyl transfer could not be observed in any conditions described under “Experimental Procedures.”NDNDThis workE124D140.170.00123000.15This workP143Q142.10.151202.4This workS150A17000.0660.000039——Ref. 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google ScholarS150C110.0570.0052——This workS150T371.30.035——This workN152A8000.0400.000050——Ref. 19Nureki O. Watanabe K. Fukai S. Ishii R. Endo Y. Hori H. Yokoyama S. Structure. 2004; 12: 593-602Abstract Full Text Full Text PDF PubMed Scopus (102) Google ScholarN152DNDNDNDThis workN152ENDNDNDThis worka Kinetic parameters for AdoMet were previously reported in the reference.b —, kinetic parameters for the transcript could not be calculated, because the affinity to AdoMet was very poor.c ND, methyl transfer could not be observed in any conditions described under “Experimental Procedures.” Open table in a new tab Fig. 2AdoHcy affinity column chromatography. The affinity of the wild type and the variant TrmH proteins for AdoHcy were tested by AdoHcy affinity column chromatography. Each protein sample (10 μg each) was loaded onto the column (Control). The column was washed by five column volumes of 50 mm KCl buffer B and combined with the flow-through fraction (Flow through). The column was washed by five column volumes of 2 m KCl buffer B (Wash). The proteins that absorbed were eluted by the addition of five column volumes of the buffer B containing 2 m KCl and 6 m urea (Elution). The protein in each fraction was collected by the trichloroacetic acid precipitation and then analyzed by 15% SDS-polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Analytical gel filtration. The subunit structures of the wild-type (A) and variant (B–E) TrmH proteins were analyzed by Superdex-75 gel filtration. Specifically, the R41A (B) and S150A (C) variants were eluted at 10.3 ml, which is slightly faster than the elution profile of the wild-type enzyme. The locations of the marker proteins are shown in the elution profile of the wild-type enzyme (A).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Biochemical Analyses—We assessed the mutant proteins for several biochemical properties. First, the kinetic parameters for both AdoMet and tRNA were determined (Table I). In this analysis, a yeast tRNAPhe transcript was used as the substrate tRNA, because this tRNA has been well characterized (13Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F.C. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (803) Google Schola" @default.
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• W2018073387 title "Roles of Conserved Amino Acid Sequence Motifs in the SpoU (TrmH) RNA Methyltransferase Family" @default.
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