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Genes to Cells (2008) 13, 807-816. doi:10.1111/j.1365-2443.2008.01207.x
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Stabilization of tRNA (m1G37) methyltransferase [TrmD] from Aquifex aeolicus by an intersubunit disulfide bond formation

 Takashi Toyooka1, Takako Awai1, Tamotsu Kanai2, Tadayuki Imanaka2 and Hiroyuki Hori1,3,*

1 Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, and
3 Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan
2 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Recombinant Aquifex aeolicus TrmD protein has a Cys20–Cys20 disulfide bond between its two subunits. This was demonstrated by SDS–polyacrylamide gel analysis of wild-type enzyme and C20S mutant protein (in which the Cys20 residue is substituted by serine), in the absence or presence of various concentrations of dithiothreitol. Analytical gel-filtration chromatography revealed that the C20S mutant protein forms a dimer structure even though it is missing the disulfide bond. Western blotting analysis suggests that the Cys20–Cys20 disulfide bond is formed in native TrmD protein in living A. aeolicus cells. Incubation at 85 °C for 20 min caused the precipitation of more than half of the C20S protein, while more than 70% of the wild-type enzyme was soluble at that temperature. This assay clearly demonstrates that the disulfide bond enhances the protein stability at 85 °C. A kinetic assay showed that the methyl-transfer activity of the C20S mutant protein was slightly less than that of the wild-type enzyme at 70 °C. Comparison of the CD-spectra of wild-type and C20S proteins reveals that some of the {alpha}-helices in the C20S mutant protein are less tightly packed than those of the wild-type enzyme at 70 °C.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
To date, more than 100 modified nucleosides have been identified in various RNA species and the majority of them have been found in tRNA molecules (Dunin-Horkawicsz et al. 2006). All of the modified nucleosides are introduced into tRNA by specific modification enzymes during the post-transcriptional process (Garcia & Goodenough-Lashhua 1998; Grosjean 2005; Grosjean et al. 2007). N1-Methylguanine at position 37 (m1G37) is found in several tRNA molecules, which have a G36G37 (Björk 1995; Sprinzl et al. 1998; Rozenski et al. 1999) or A36G37 (Kimball et al. 1974) sequence. The m1G37 does not form a Watson–Crick base pair with C in mRNA and prevents frame-shift error (Björk et al. 1989). Thus, this modification contributes to maintenance of the reading frame during the translation process on the ribosome (Hagervall et al. 1993; Farabaugh & Björk 1999). This is consistent with the fact that the m1G37 modification is essential for growth in several eubacteria such as Escherichia coli (Baba et al. 2006), Salmonella typhimurium (Björk et al. 1989), and Streptococcus pneumoniae (O'Dwyer et al. 2004). The m1G37 modification is found in tRNA molecules from three domains of life (Björk et al. 2001) as well as organelle (chloroplast and mitochondria) (Osorio-Almeida et al. 1980; Lee et al. 2007). Further, the first step of wybutosine (or related analogs) formation at position 37 in eukaryotic or archaeal tRNAPhe is m1G37 modification (Droogmans & Grosjean 1987; Noma et al. 2006; Suzuki et al. 2007). The methylation of the N1 atom of G37 is catalyzed by tRNA (m1G37) methyltransferase [EC 2. 1. 1. 31] (Garcia & Goodenough-Lashhua 1998). In eubacteria, tRNA (m1G37) methyltransferase is encoded by the trmD gene (Byström & Björk 1982). In contrast, the eukaryotic, archaeal and yeast mitochondrial enzyme are encoded by the trm5 gene (Christian et al. 2004; Lee et al. 2007). Both TrmD and Trm5 enzymes belong to the S-adenosyl-L-methionine (AdoMet) dependent methyltransferases, although the protein folds are expected to be considerably different from each other (Christian et al. 2004). TrmD proteins have a distinct topological knot (Ahn et al. 2003; Elkins et al. 2003; Tkaczuk et al. 2007) (the so-called "trefoil knot" (Nureki et al. 2002, 2004)) structure and the fold is classified as Class IV (Schubert et al. 2003). In contrast, Trm5 proteins contain typical motifs conserved among Rossman fold methyltransferases (Christian et al. 2004), strongly suggesting that they have a Class I fold (Schubert et al. 2003).

For several years, we have studied the RNA modification enzymes from Aquifex aeolicus (Hori et al. 2003; Okamoto et al. 2004; Takeda et al. 2006; Tomikawa et al. 2008). Aquifex aeolicus is a hyper-thermophilic eubacterium (Burggraf et al. 1992; Deckert et al. 1998). This eubacterium was isolated from boiling water in a hot spring in Yellowstone National Park. Although the oxygen concentration in boiling water is very low compared to cold water, A. aeolicus lives in the presence of oxygen. In such conditions, cysteine residues in proteins are expected to be rapidly oxidized. Recently, the crystal structure of A. aeolicus TrmD was solved (Liu et al. 2003) (Fig. 1A). All TrmD proteins reported thus far form a homodimer structure (Ahn et al. 2003; Elkins et al. 2003; Liu et al. 2003). This is consistent with a proposed tRNA docking model, in which rigid binding of tRNA requires two subunits (Ahn et al. 2003). The A. aeolicus TrmD was expected to be in oligomeric (dimer or trimer) state in solution, because three monomers were identified in an asymmetric unit of the crystal (Liu et al. 2003). In a series of the studies on RNA modification enzymes from A. aeolicus (Hori et al. 2003; Okamoto et al. 2004; Takeda et al. 2006; Tomikawa et al. 2008), we have reported the substrate RNA recognition mechanism of A. aeolicus TrmD protein (Takeda et al. 2006). During the course of the purification, we found the presence of a disulfide bond(s) between the two subunits. In the crystal structure, the Cys20 residues in the two subunits are located at a distance of 3.54 Å and the residues do not form a disulfide bond (Liu et al. 2003). However we considered that the Cys20 residues have a potential to form a disulfide bond at high temperatures in the presence of oxygen. In this article, we demonstrate formation of a disulfide bond between TrmD subunits in living A. aeolicus cells and clarify the role of the disulfide bond by biochemical studies.


Figure 1
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  		Figure 1  Dimer structure of A. aeolicus TrmD and locations of Cys residues. (A) Dimer structure of A. aeolicus TrmD. The different subunits (monomer1 and monomer2) are represented in yellow and cyan, respectively. Cys20 and Cys114 residues are in red and green, respectively. The dotted line shows the expected disulfide bond between the two Cys20 residues. (B) Amino acid sequence alignment of TrmD proteins. This alignment was generated by Clustal W 1.83 (Thompson et al. 1994) and ESPript (Gouet et al. 1999) programs. The conserved and semi-conserved residues are enclosed and highlighted in red, respectively. Secondary structures are shown at the top of the alignment: arrows, coils, and Ts mean β-sheets, helices, and turns, respectively. The positions of Cys20 and Cys114 are shown at the bottom of the alignment by red and green triangles, respectively. The sources of the TrmD proteins are shown at the left side in abbreviated form: AQUAE, A. aeolicus; THEMA, Thermotoga maritima; ESCCO, Escherichia coli; HAEIN, Haemophilus influenzae; STRPN, Streptococcus pneumoniae; SYNEC, Synechocystis sp.; TREPA, Treponema pallidum; MYCPN, Mycoplasma pneumoniae. The polypeptide lengths are given at the end of the alignment.

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Possibility of disulfide bond formation between two subunits

Figure 1A shows the crystal structure of A. aeolicus TrmD (PDB code: 1oy5) (Liu et al. 2003). Although the coordinates of three monomers have been deposited in the database, we depict the figure as a dimeric form based on reasons as follows. Liu et al. have predicted that the dimer structure is a functional form of the A. aeolicus TrmD (Liu et al. 2003). Our SDS-PAGE and gel-filtration analyses in the current study provide experimental verifications for their prediction (see Figs 2A and 3A). Thus, their crystal structure and our biochemical studies are in good agreement with the concept that the functional form of the A. aeolicus TrmD is a dimer. This is in line with structures of the other TrmD proteins (Ahn et al. 2003; Elkins et al. 2003). Furthermore, it should be mentioned that our biochemical experiments in the current study support the predicted dimer interface in the crystal study (Liu et al. 2003). Because amino acid residues 165–178 and the C-terminal region (22 amino acids) are not seen in the crystal structure, we cannot judge whether A. aeolicus TrmD has a topological knot like the other TrmD proteins (Ahn et al. 2003; Elkins et al. 2003). However high conserved amino acid sequence among TrmD proteins (Fig. 1B) strongly suggest that A. aeolicus TrmD has a topological knot. Aquifex aeolicus TrmD protein has two cysteine residues per subunit (Cys20 and Cys114) (Fig. 1). Although Cys114 is conserved among TrmD proteins (Fig. 1B), a systematic site-directed mutagenesis study on E. coli TrmD enzyme revealed that alanine substitution of this residue does not have an obvious effect on the methyl-transfer activity (Elkins et al. 2003). In contrast, the Cys20 residue is not conserved and is A. aeolicus TrmD specific. The Cys20 residue is located on the {alpha}1-helix, which forms a dimer interface. The two Cys20 residues in the dimer structure are located at a distance of 3.54 Å (Fig. 1A). Based on these observations, we considered that the two Cys20 residues might form a disulfide bond between the two subunits at high temperatures under atmosphere.


Figure 2
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  		Figure 2  Disulfide bond formation between two subunits in the wild-type TrmD protein and absence of the disulfide bond in the C20S mutant protein. (A) The wild-type protein (1 µg each) was analyzed by 15% SDS-PAGE in the absence (lane 0 mM) or presence of various concentrations (lanes 2.5, 5, 10, 15, 20, and 25 mM) of DTT. Arrows show the positions of the dimmeric and monomeric forms. The gel was stained with Coomassie brilliant blue. (B) The C20S mutant protein (1 µg each) was analyzed by the same method as described in panel (A).

 
Recombinant TrmD contains a disulfide bond between the two Cys20 residues

To confirm this idea, we performed 15% SDS-PAGE in the absence or presence of various concentrations of DTT (Fig. 2A). In the absence of DTT, recombinant TrmD protein migrates at 65 kDa even after boiling for 5 min in the presence of 0.1% SDS, that is, the mobility corresponds to a dimeric form (Fig. 2A lane 0 mM). With increasing DTT concentration, a band coresponding to monomeric form appears with increasing intensity (Fig. 2A lanes 2.5–25 mM). This result clearly shows that the recombinant TrmD contains a disulfide bond(s) between the two subunits. To confirm whether the Cys20 residue is responsible for the disulfide bond formation, we made a mutant protein C20S, in which Cys20 was substituted by serine. The C20S protein was purified to homogeneity as assessed by 15% SDS-PAGE (Fig. 2B). Boiling in SDS sample buffer results in C20S protein dissociating to monomeric subunits even in the absence of DTT (Fig. 2B lane 0 mM). The concentration of DTT did not affect the protein mobility on the gel (Fig. 2B lane 2.5–25 mM). Thus, these results demonstrate that the Cys20 residue is responsible for the disulfide bond formation between the two subunits. Furthermore, we checked the subunit structure of the C20S mutant protein by analytical gel-filtration chromatography (Fig. 3). The C20S mutant protein (Fig. 3B) eluted at the same point as the wild-type protein (Fig. 3A), demonstrating that C20S mutant protein forms a dimeric structure and that the dissociated monomeric form of C20S mutant protein seen in Fig. 2B is generated by boiling in the presence of 0.1% SDS. The dimeric structure is consistent with the fact that C20S mutant protein has methyl-transfer activity as described in the section below.


Figure 3
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  		Figure 3  Analytical gel-filtration chromatography of the wild-type and C20S mutant proteins. (A) 50 µg of the wild-type enzyme was analyzed by Superdex-75 gel-filtration column chromatography. Elution positions and molecular weights of standard proteins are shown by arrows. (B) 50 µg of the C20S mutant protein was analyzed under the same condition as described in panel (A).

 
TrmD protein from living Aquifex aeolicus cells contains a disulfide bond

Purified recombinant TrmD contains a disulfide bond between the two Cys20 residues. However, we suspected that this form might be an artificial form because the purification was carried out under atmospheric condition with high protein concentration (the purified TrmD was concentrated to 17.1 mg/mL before storage) as compared to the natural condition in living cells. Therefore, we undertook to investigate the native form of A. aeolicus TrmD protein in living cells. The culture source of A. aeolicus and medium were kindly provided from Dr Harald Huber (Universitat Regensburg, Germany). We prepared anti-A. aeolicus TrmD polyclonal antibody for Western blotting detection. The cultured cells were directly resuspended in SDS-sample buffer in the absence or presence of DTT, disrupted and immediately analyzed by 12.5% SDS-PAGE (Fig. 4A): Fig. 4A lanes 1 and 2 show the Coomassie brilliant blue (CBB) staining patterns with and without DTT, respectively. As shown in Fig. 4A lanes 1 and 2, high molecular weight proteins could be observed in the absence of DTT. This result suggests that many proteins in the A. aeolicus cells have a disulfide bond(s). Figure 4A lanes 3 and 4 show the purified recombinant TrmD with and without DTT, respectively. To detect the TrmD protein in the crude extracts, we performed Western blotting analysis (Fig. 4B). As shown in Fig. 4B lane 1, native TrmD protein migrated as a dimer in the absence of DTT. In contrast, in the presence of DTT it migrated as a monomer (Fig. 4B lane 2). These results strongly suggest that natural TrmD protein in living cells contains a disulfide bond between the two subunits.


Figure 4
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  		Figure 4  Analysis of formation of disulfide bond between the two subunits of native TrmD protein in living cells by western blotting. (A) Cultured A. aeolicus cells were disrupted in SDS-sample buffer in the absence (lane 1) or presence of 100 mM DTT (lane 2) and then immediately analyzed by 12.5% SDS-PAGE. Lanes 3 and 4 show the mobilities of the purified recombinant TrmD in the absence (lane 3) and presence (lane 4) of 100 mM DTT. The gel was stained with Coomassie brilliant blue. (B) The TrmD protein in the cell extract was detected by western blotting analysis. The SDS-PAGE was performed under the same condition as described in panel (A) and then electro-blotting was performed. The first antibody (anti-A. aeolicus TrmD polyclonal antibody) was customized. The fluorescence derived from secondary antibody was monitored on a Typhoon model 9410 system.

 
Cys20–Cys20 disulfide bond contributes to protein stability at 85 °C

To address the function of the Cys20–Cys20 disulfide bond, we incubated wild-type enzyme and C20S mutant protein at various temperatures (25, 55, 70, 80, and 85 °C). Below 80 °C, both proteins were soluble (data not shown). However, some portion of protein began to precipitate at 85 °C (Fig. 5). In Fig. 5, TS, S, and P represent total sample without heat treatment, soluble fraction and precipitated fraction, respectively. In the case of wild-type enzyme, incubation for 20 min at 85 °C caused approximately 30% of the protein to be precipitated (Fig. 5 left). In contrast, more than half of the C20S mutant protein precipitated under the same conditions (Fig. 5 right). These results clearly show that the Cys20–Cys20 disulfide bond contributes to protein stability at 85 °C.


Figure 5
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  		Figure 5  Heat-stability of wild-type and C20S mutant proteins. Wild-type (1 µg) or C20S mutant (1 µg) protein were incubated at 85 °C for 0 (TS), 10, or 20 min and then centrifuged to separate the soluble (S) and precipitated (P) fractions. The samples were analyzed by 15% SDS-PAGE and Coomassie brilliant blue staining.

 
Kinetic study

We considered that the difference in protein stability between wild-type and C20S mutant proteins may have an effect on the methyl-transfer activity. However, because a portion of the protein precipitated at 85 °C, we measured initial velocities at 55 and 70 °C. The initial velocity of the C20S mutant protein at 70 °C was slightly slower than that of the wild-type enzyme (data not shown). In contrast, the velocity of the mutant protein at 55 °C was comparable to that of the wild-type enzyme (data not shown). We checked the initial velocities of the wild-type and C20S proteins more than ten times at 55 and 70 °C, and confirmed the reproducibility. After these pilot experiments, we determined kinetic parameters both for AdoMet and tRNA transcript (Table 1). As shown in Table 1, there are slight but clear differences between the kinetic parameters of wild-type and C20S mutant enzymes with the activity of C20S mutant enzyme being slightly lower at 70 °C. We thought it possible that this phenomenon might be caused by a change in the stability of the dimer interface at 70 °C. However, it is noteworthy that the substrate tRNA used in this assay was A. aeolicus tRNAPro transcript. Because the stem structures in this transcript are mainly formed by G–C base pairs, the melting temperature of this transcript is expected to be more than 80 °C. However, the local structure of this transcript seems to be loosened at 70 °C: the relative Vmax/Km values of both enzymes at 55 °C are larger than those at 70 °C through an increase in Km values for tRNA. Therefore, in this case, temperature-dependence of fine structure of the substrate tRNA should be taken into consideration. Thus, we found small but clear temperature-dependent differences in the kinetic parameters (initial velocities) between the wild-type and C20S proteins. However the difference is too small to allow us to conclude that it is solely due to the presence or absence of the Cys20–Cys20 disulfide bond. At high temperatures (> 85 °C), the Cys20–Cys20 disulfide bond will contribute to the activity through stabilization of the subunit–subunit interaction.


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  		Table 1  Kinetic parameters for AdoMet and A. aeolicus tRNAPro transcript
 
CD-spectra measurements

The experimental results described above prompted us to measure the CD-spectra of the wild-type and C20S proteins at 25, 55, and 70 °C. Figure 6A shows the CD-spectra of the wild-type TrmD protein. With increasing temperature, the valley in the spectrum observed at 220 nm becomes shallower but it still clearly present. In general, this valley reflects the state of {alpha}-helices in the protein (Clark et al. 1988). The {alpha}-helical content of the wild-type enzyme at 70 °C is calculated to be 25% (Greenfield 1996). In contrast, the CD-spectra of the C20S mutant protein show that at 70 °C the valley at 220 nm is lost (Fig. 6B): the calculated {alpha}-helix content of the mutant protein at 70 °C is 23%. These results show that at 70 °C a portion of {alpha}-helices in the C20S mutant protein is more loosened than in the wild-type enzyme, although the C20S mutant protein retains sufficient activity at that temperature.


Figure 6
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  		Figure 6  CD-spectra of the wild-type and C20S mutant TrmD proteins at 25, 55, and 70 °C. Solid, broken, and dotted lines show the spectra at 25, 55, and 70 °C, respectively. The calculated {alpha}-helical content at each temperature is given in the figure. (A) CD-spectra of the wild-type TrmD protein. (B) CD-spectra of the C20S TrmD protein.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we demonstrate protein stabilization at high temperature (85 °C) by disulfide bond formation between two subunits of A. aeolicus TrmD enzyme. Although the detailed enzyme mechanism of TrmD has not been clarified, efficient turn over of the catalytic cycle probably requires the interaction of two subunits. Ahn et al. have proposed a tRNA docking model for Haemophilus influenzae TrmD dimer (Ahn et al. 2003). In their model, the anticodon arm in tRNA is captured by one subunit, and the variable region and acceptor-stem are in contact with another subunit. In our previous work, we reported that A. aeolicus TrmD can methylate the anticodon arm micro-helix RNA (Takeda et al. 2006). More recently, Christian et al. have reported that E. coli TrmD catalyzes a methyl-transfer to hairpin structure including an anticodon arm (Christian & Hou 2007). Although these studies have revealed that the anticodon arm structure is a minimum required element, the velocities of methyl-transfer to these transcripts are slow: the rigid binding of tRNA is probably required for efficient turn over of the catalytic cycle. Furthermore, in the case of TrmH (Persson et al. 1997; Hori et al. 2002) (tRNA (Gm18) methyltransferase), which is classified into the SPOUT super-family of RNA methyltransferases together with TrmD (Anantharaman et al. 2002; Tkaczuk et al. 2007), the interaction of two subunits is required for release of the product (S-adenosyl-L-homocysteine) (Watanabe et al. 2005, 2006). Thus, the interaction of two subunits interaction is important for these SPOUT RNA methyltransferases.

In general, cysteine is rapidly oxidized at high temperatures in the presence of molecular oxygen. Therefore, several aerobic thermophilic bacteria avoid usage of Cys residues in protein. For example, the cysteine content of proteins from Thermus thermophilus, an extreme-thermophilic eubacterium, is low compared to that from mesophiles (Henne et al. 2004). However, recent studies demonstrated the existence of different scenarios, in which disulfide bonding enhances protein stability at high temperatures (Beeby et al. 2005). In their computational work, it has been predicted that disulfide bond richness in intracellular proteins is widespread in a subset of thermophiles including A. aeolicus. Indeed, our analysis of the A. aeolicus crude extract shows that many proteins have a disulfide bond(s) (Fig. 4A). Further, in the case of tRNA (m1A57, 58) methyltransferase [Archaea TrmI] from Pyrococcus abyssi, it has been reported that four disulfide bonds stabilize the tetramer structure at 85 °C (Roovers et al. 2004). These studies combined with our own show that some thermophiles utilize disulfide bonds to stabilize protein structure at high temperatures. Further, the existence of a protein disulfide oxidoreductase in A. aeolicus has been reported recently (Pedone et al. 2006). Thus, A. aeolicus may utilize disulfide bond formation actively.

The Cys20 residue in TrmD of A. aeolicus is not conserved among the other TrmD proteins. However, an amino acid sequence alignment shows that the residue is substituted by a bulky hydrophobic amino acid residue in the TrmD proteins from thermophiles (Fig. 1B). For example, the residue is substituted by valine in Thermotoga maritima TrmD. This observation suggests that the consolidation of hydrophobic interaction(s) is an alternative strategy for stabilization of subunit structure instead of disulfide bond(s) formation. Further, we demonstrate that the C20S mutant enzyme catalyzes the methyl-transfer reaction at a slightly slower rate than the wild-type enzyme at high temperature (70 °C). This difference maybe explained by the results of the CD-spectra measurement which shows that a portion of the {alpha}-helices in the C20S mutant protein is more loosened than in wild-type enzyme at 70 °C. This result may suggest that the local flexibility at the subunit interface is required for efficient methyl-transfer.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

[Methyl-14C]-AdoMet (1.95 GBq/mmol) and [methyl-3H]-AdoMet (2.47 TBq/mmol) were purchased from ICN. Cold AdoMet was obtained from Sigma. Other chemical reagents were of analytical grade.

Purification of recombinant TrmD proteins

Recombinant TrmD was expressed in E. coli cells and purified to homogeneity as assessed by 15% SDS-polyacrylamide gel electrophoresis (PAGE) as reported previously (Takeda et al. 2006). The substitution of Cys20 by Ser was performed using the Quick change mutagenesis kit (Stratagene). The DNA sequences were analyzed on ABI PRISM 310 DNA sequencers. The purification of the mutant protein (C20S) was performed using the same method as for the wild-type enzyme. The quantity of protein was measured with the Bio-Rad protein assay kit using bovine serum albumin as the standard. The purified proteins were mixed with glycerol (final concentration 50%), and stored at –30 °C. To confirm the Cys20–Cys20 disulfide bond formation, we analyzed the wild-type and C20S mutant proteins in the absence or presence of various concentrations of dithiothreitol (DTT) as follows. The purified samples (wild-type and C20S mutant proteins) were dialyzed against buffer A (50 mM Tris–HCl (pH 7.5), 6 mM MgCl2 and 50 mM KCl). An amount of 50 µL of the sample (10 µg) (DTT final concentrations of 0, 2.5, 5, 10, 15, 20, or 25 mM) and 50 µL of SDS-PAGE sample buffer (50 mM Tris–HCl (pH 6.8), 2% SDS, 0.2% bromophenol blue, and 10% glycerol) were mixed, boiled for 5 min and then analyzed by 15% SDS-PAGE.

Analytical gel-filtration

Analytical gel filtration was performed on an ÄKTAprime chromatography system (GE healthcare) equipped with a Superdex 75 column (10/30; column volume, 23.6 mL) at room temperature. The column was first equilibrated with buffer B (50 mM Tris–HCl (pH7.6), 5 mM MgCl2, 6 mM 2-mercaptoethanol, 200 mM KCl), and the sample was then injected. The flow rate was 0.5 mL/min. Elution profiles were monitored by the absorption of UV at 280 nm.

Culture of Aquifex aeolicus

The culture source of Aquifex aeolicus and 100 mL of culture medium in controlled gas (H2–CO2 (v/v, 4 : 1) mixed gas pressurized by air to 2 atm) were kindly provided by Dr Harald Huber (Universitat Regensburg, Germany). The culture was performed at 85 °C for 24 h.

Western blotting

Customized rabbit anti-A. aeolicus TrmD serum was prepared by Kitayama Labes Co., Ltd, Japan. The polyclonal antibody fraction was partially purified using the Econo-pac serum IgG purification kit (Bio-Rad). Cultured cells were directly resuspended in SDS-PAGE sample buffer, and then disrupted. A half of sample was added DTT to a final concentration of 100 mM. The samples with and without DTT were boiled and immediately loaded onto a 12.5% SDS-polyacrylamide gel. Electro-blotting to a PVDF membrane (Immobilon transfer membrane IPVH00010, pore size 0.45 µm, Millipore) was performed using a semi-dry blotting system (NA-1515B, Nippon Eido) according to the manufacturer's instructions. TrmD protein was detected using Alexa Fluor 488 anti-rabbit IgG (Invitrogen) as a secondary antibody and visualized using a Typhoon model 9410 (GE healthcare).

Heat stability assay

The concentration of wild-type enzyme or C20S mutant protein was adjusted to 60 µg/mL in 20 µL of buffer C (50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 50 mM KCl, and 10 mM EDTA). The samples were incubated for 0, 10, or 20 min at 85 °C and then centrifuged at 12 000 g for 15 min. The pellets were resuspended in 20 µL of buffer C and analyzed by 15% SDS-PAGE together with the supernatants. The band intensities were measured by NIH image version 1.63.

Kinetic parameters

Aquifex aeolicus tRNAPro transcript was prepared as reported previously (Takeda et al. 2006) and purified by 10% polyacrylamide gel (7 M urea) electrophoresis. Kinetic parameters of the wild-type and C20S mutant enzymes with AdoMet and tRNA were determined at 55 and 70 °C according to our previous report (Takeda et al. 2006).

CD-spectra measurements

CD spectra were measured on a JASCO J-820 spectropolarimeter equipped with a JASCO PTC-423L thermo-controller. Cuvettes with a 1 mm path length were used. Each sample (350 µg/mL) in buffer A was pre-incubated at 25, 55, or 70 °C for 20 min and then the spectrum recorded from 300 to 200 nm at each temperature. The scan speed was 100 nm/min. The spectra shown in this article are the average of three scans.


   Acknowledgements
 
Authors thank Professor Yaeta Endo (Ehime University) for use of laboratory facilities, Dr Kazunori Watanabe (Ehime University) for advice regarding the kinetic study and CD spectra measurement, and Miss Chikako Iwashita (Ehime University) for technical support in Western blotting analysis. This work was partly supported by a Grant-in-aid (18048032) for Science Research on Priority Areas, and a Grant-in-aid (17613003, 19350087) for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshikazu Nakamura

* Correspondence: hori{at}eng.ehime-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Ahn, H.J., Kim, H.W., Yoon, H.J., Lee, B., Suh, S.S. & Yang, J.K. (2003) Crystal structure of tRNA (m1G37) methyltransferase: insights into tRNA recognition. EMBO J. 22, 2593–2603.[CrossRef][Medline]

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Received: 21 February 2008
Accepted: 30 April 2008





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