Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans

Protein Expression and Purification 88 (2013) 13–19

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Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans Yuichiro Nomura a, Yumiko Onda a, Satoshi Ohno a, Hiroki Taniguchi b, Kaori Ando b, Natsuhisa Oka b, Kazuya Nishikawa a, Takashi Yokogawa a,⇑ a b

Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Department of Chemistry, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

a r t i c l e

i n f o

Article history: Received 31 August 2012 and in revised form 14 November 2012 Available online 29 November 2012 Keywords: ArcTGT Archaeosine 7-Deazaguanine Methanosarcina acetivorans

a b s t r a c t Many archaeal tRNAs have archaeosine (G+) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G+ biosynthesis, archaeosine tRNAguanine transglycosylase (ArcTGT)1 catalyzes the base exchange reaction from guanine to 7-cyano-7deazaguanine (preQ0). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ0 at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G+ biosynthesis. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Transfer RNAs contain a large number of modified nucleosides, which may play a role in the efficiency and fidelity of protein biosynthesis. In the past, various modified nucleosides have been identified from all three kingdoms of life, and some nucleosides are specifically conserved within a kingdom. In particular, 7-deazaguanine derivatives are interesting molecules because this modification is caused by a base exchange reaction [1], whereas the majority of modifications are caused by addition of functional groups to a base. In Bacteria and Eucarya, queosine (Q) [2] is found at position 34 of tRNATyr, tRNAHis, tRNAAsn and tRNAAsp, which have GUN anticodons [3]. This modification from G to Q is thought to be involved in strengthening the base pairing between codon and anticodon [4,5]. In contrast, many archaeal tRNAs have archaeosine

⇑ Corresponding author. Fax: +81 58 293 2794. E-mail address: [email protected] (T. Yokogawa). ArcTGT, archaeosine tRNA-guanine transglycosylase; QueTGT, tRNA-guanine transglycosylase; PCR, polymerase chain reaction; MOPS, 3-[N-morpholino]propanesulfonic acid; DTT, dithiothreitol; iPCR, inverse PCR; IPTG, isopropylthiogalactoside; TCA, trichloroacetic acid; HIFP, 1,1,1,3,3,3-Hexafluoro-2-propanol; TEA, triethylamine. 1

1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.11.009

(G+) [6] at position 15 (Fig. 1), which may tighten up the tertiary base pair, G15–C48 [7]. In Bacteria, the 7-deazaguanine derivative, preQ1 [8], is incorporated by a tRNA-guanine transglycosylase (QueTGT) [9], and then preQ1-tRNA is converted to Q-tRNA by the subsequent actions of enzymes QueA and QueG [10,11]. In Eucarya, Q is directly introduced to tRNA through a salvage pathway [12]. In Archaea, ArcTGT, which is a homologue of the bacterial QueTGT, catalyzes the base exchange reaction from G15 to preQ015 [13]. Recently, several enzymes have been reported on the basis of genome informatics that may be involved in conversion from preQ0-tRNA to G+-tRNA [14,15], but the mechanism of this reaction is still unclear. There exist two subclasses in ArcTGT: the full-size and the split types. Studies in the full-size type are more advanced because the crystal structure of Pyrococcus horikoshii ArcTGT has already been determined [16,17]. The structure of a full-size ArcTGT can be roughly divided into the N-terminal and C-terminal domains from the crystal structure. The N-terminal domain contains catalytic residues for the base exchange reaction and the Cterminal domain has a binding site for the acceptor stem of tRNA [16–20]. On the other hand, a split ArcTGT appears to consist of two peptides, corresponding to the N-terminal or C-terminal domains of a full-size ArcTGT. Both full-size and spilt types are found

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Y. Nomura et al. / Protein Expression and Purification 88 (2013) 13–19

Fig. 1. Biosynthetic pathway of archaeosine.

in the genomes of Euryachaeota but only the split type has been found to date in Crenarchaeota genomes [14,15]. Therefore, the split type is more general in Archaea than the full-size type. Nevertheless, native split ArcTGT has not been isolated from cell extracts, so there have been few studies on split ArcTGT. In early work, Watanabe et al. claimed to have isolated ArcTGT as a single peptide from Haloferax volcanii cell extract [13]. However, H. volcanii ArcTGT is only assumed to be split according to its genome information. In addition, Sabina et al. reported that only the N-terminal domain of the full-size ArcTGT could precisely recognize position 15 of tRNA [21]. Consequently, the C-terminal domain may not be necessary for substrate recognition. In other words, it is not clear that the two peptides assumed to form a complex are actually subunits of ArcTGT. To resolve this, it is important to purify native ArcTGT from archaeal species encoding split ArcTGT. Here, we report the isolation of native ArcTGT from M. acetivorans and show that two peptides formed a robust complex in cells. Moreover, recombinant ArcTGT with a histidine tag was prepared by co-overexpressing two peptides in E. coli. Finally, we were able to prepare preQ0-tRNA with this recombinant ArcTGT.

Materials and methods General Synthetic DNA oligomers were obtained from Operon Biotechnologies. [8-14C] -guanine hydrochloride was purchased from Moravek Biochemicals (Brea, USA). M. acetivorans C2A was sourced from Riken BRC (Tsukuba, Japan). DNA polymerases used for polymerase chain reaction (PCR) were obtained from Takara Bio (Otsu, Japan). T4 DNA ligase was obtained from Nippon Gene (Toyama, Japan). Restriction endonucleases were obtained from Fermentas (Burlington, Canada). Ni-NTA Superflow column was purchased from QIAGEN (Tokyo, Japan). Resins for column chromatography, apart from Ni-NTA Superflow resin, and T4 polynucleotide kinase were obtained from GE Healthcare (Buckinghamshire, UK). PreQ0 was synthesized following a procedure reported in the literature [22].

Organism and growth conditions M. acetivorans C2A [23] was cultivated under strictly anaerobic conditions at 37 °C in DSM medium 304 with the following modifications. 10 ml of vitamin solution from DSM medium 141 was substituted for yeast extract and Na2CO3 was replaced with 20 mM 3-[N-morpholino]propanesulfonic acid (MOPS), pH 7.0, as the buffer. Cultures were grown for 4 days in 4 L of this modified DSM medium in a CULTURE BAG system (obtained from Fujimori Kogyo Co., Ltd., Tokyo, Japan).

Purification of native ArcTGT M. acetivorans C2A cells (30 g) were suspended in 75 ml of buffer A (20 mM Tris–HCl (pH 7.6), 1 mM MgCl2, 5 mM DTT) containing 40 mM KCl and disrupted by sonication. An S100 extract was obtained after centrifugation at 100,000  g for 4 h. The pellet from sequential precipitation of proteins at 50% and 70% ammonium sulfate saturation was dissolved in 39 ml of buffer A containing 1.2 M (NH4)2SO4, applied to a Phenyl Sepharose HP column (20 ml, GE Healthcare), and eluted with a linear gradient (400 ml) from 1.2 to 0.3 M (NH4)2SO4 at a flow rate of 2.0 ml/ min, with 20 ml fraction collection. The active fractions were concentrated with an Amicon Ultra-15 10 K filter device (Merck Millpore, Darmstadt, Germany), and applied to a HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare) with buffer A containing 50 mM KCl at a flow rate of 0.8 ml/min, with 4 ml fraction collection. The active fractions were applied to a HiTrap Q HP column (5 ml, GE Healthcare). Proteins were eluted with a linear gradient (100 ml) from 50 to 300 mM KCl at a flow rate of 1.0 ml/min, with 5 ml fraction collection. The active fractions were dialyzed against buffer B (20 mM HEPES–KOH (pH 7.0), 1 mM MgCl2, 40 mM KCl, 5 mM dithiothreitol (DTT)). The dialysate was applied on a HiTrap Heparin HP column (1 ml, GE Healthcare), and eluted with a linear gradient (20 ml) from 40 to 600 mM KCl at a flow rate of 0.5 ml/ min, with 1 ml fraction collection. The buffer in active fractions was exchanged for buffer A containing 40 mM KCl with an Amicon Ultra-15 10 K filter device. The pool was applied to a Mono Q column (1 ml, GE Healthcare), and eluted with a linear gradient (20 ml) from 40 to 300 mM KCl at a flow rate of 0.5 ml/min, with 1 ml fraction collection. Finally, the active fractions were concentrated with an Amicon Ultra-0.5 10 K filter device. The amount of ArcTGT in the fraction of the Mono Q column was estimated by the density of bands on SDS–PAGE using ImageJ software (http:// rsb.info.nih.gov/ij/). The other amount of the protein at each step was measured by a previously described method [24]. Construction of protein expression vectors The protein expression vector, pETY_Blue, is a high-copy-number plasmid adaptable for the blue/white screen. This was constructed in our laboratory by the following procedure. A DNA fragment including the lac promoter and the sequence corresponding to the b-galactosidase a fragment was amplified using the E. coli Q13 strain with the forward primer 5’-GGG GAA GCT TGC GCA ACG CAA TTA ATG-3’ and the reverse primer 5’-GGG GGC TAG CTT ATT CGC CAT TCA GGC-3’, and was inserted into the region between the Hind III and Nhe I sites of pET21a(+) (Merck Millipore). The transformant with the resulting plasmid, named pET_Azure, formed blue colonies on a plate containing X-Gal, IPTG and ampicillin. Then, the whole region of pET_Azure without the gene for RNA I inhibition modulator protein was amplified by the

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inverse PCR (iPCR) technique [25] with primers 5’-GGC GCT CTT CCG CTT CCT CGC-3’ and 5’-GGT GCC TAA TGA GTG AGC TAA C3’. The purified PCR product was circularized by treating with T4 polynucleotide kinase and T4 DNA ligase. Finally, the RNA I gene was mutated by the QuikChangeÒ method with the primers 5’GGC TAC ACT AGA AGA ACA GTA TTT GGT ATC-3’ and its reverse complement. M. acetivorans ArcTGT is thought to consist of two peptides (GenBank Accession No. AE010299). One is the product of MA4419 and the other is that of MA0121. MA4419 was amplified by PCR with the forward primer 5’-GGG AAT TCA TAT GTC AGC GAT ATT TGA AA-3’ and the reverse primer 5’-GGG GGT CGA CTT CTT TTT TCC AGG CTG-3’, while MA0121 was amplified with 5’GGG AAT TCA TAT GAA CAG CAA TGT GGA AA-3’ and 5’-TGT TCG ACT TAA GCA CTC GAG TTA AAT TAA GAT TTT ATG ACT TA-3’ from M. acetivorans C2A genomic DNA. MA4419 digested with Nde I and Xho I or MA0121 digested with Nde I and Sal I was inserted into the region between Nde I and Xho I of pETY_Blue, then designated pETY-MA4419 or pETY-MA0121, respectively. The co-expression vector for MA4419 and MA0121 was constructed using the In-FusionÒ Advantage PCR Cloning system (Clontech, Otsu, Japan). To prepare vector DNA, the pETYMA4419 sequence was amplified by iPCR with 5’-TGC TTA AGT CGA ACA CGT AGA GGA TCG AGA-3’ and 5’-CGC ATC GTG GCC GGC ATC AC-3’. To prepare the insert DNA, MA0121 was amplified by PCR with 5’-GCC GGC CAC GAT GCG TAA TAC GAC TCA CTA TAG GG-3’ and 5’-TGT TCG ACT TAA GCA CTC GAG TTA AAT TAA GAT TTT ATG ACT TA-3’. Both PCR products were ligated using the InFusion reaction. The resulting plasmid was named pETYMA0121-MA4419-His6. Overexpression and purification of recombinant ArcTGT E. coli ER2566 harboring pETY-MA0121-MA4419-His6 was cultivated at 37 °C shaking at 140 rpm in 100 ml of LB medium supplemented with 50 lg/ml ampicillin. The proteins expression was induced by adding isopropylthiogalactoside (IPTG) at a final concentration of 0.5 mM when A600 reached 0.7. The cells were then cultivated at 37 °C for 4 h. The cells were harvested by centrifugation at 5800  g for 15 min, re-suspended by 10 ml of buffer C (20 mM Tris–HCl (pH 7.6), 1 mM MgCl2, 5 mM DTT, 200 mM KCl), and disrupted by sonication. The supernatant obtained by centrifugation at 30,000  g for 30 min was loaded onto a Ni-NTA Superflow column (1 ml), and then eluted with a linear gradient (40 ml) from 0 to 300 mM imidazole at a flow rate of 0.5 ml/min, with 1 ml fraction collection. The fractions containing recombinant proteins on SDS–gel were concentrated with an Amicon Ultra-15 10 K filter device. The concentrated pool was applied onto a Superdex 200 column (GE Healthcare) and eluted with buffer A containing 50 mM KCl at a flow rate of 0.25 ml/min, with 1 ml fraction collection. The fractions containing recombinant proteins were applied onto the HiTrap Q HP column (1 ml) and eluted with a linear gradient (20 ml) from 0 to 300 mM KCl at a flow rate of 0.5 ml/min, with 1 ml fraction collection. The buffers in the fractions containing recombinant proteins were exchanged with buffer A containing 100 mM KCl using an Amicon Ultra-4 10 K filter device. The resulting pool was applied onto the HiTrap Heparin HP column (1 ml) and eluted with a linear gradient (20 ml) from 250 to 550 mM KCl at a flow rate of 0.5 ml/min, with 1 ml fraction collection. The fractions containing only recombinant proteins were concentrated and the buffer in the fractions was exchanged with buffer A containing 40 mM KCl using an Amicon Ultra-4 10 K filter device. The amount of the isolated recombinant ArcTGT was determined from the absorbance at 280 nm and ProtParam tool (http:// web.expasy.org/protrapam/). The other amount of the protein at each step was measured by a previously described method [24].

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Preparation of M. acetivorans tRNAMet transcript A DNA fragment including the T7 promoter and pre-tRNAMet (MAt4696) was amplified by PCR with the forward primer 5’GGG GGA ATT CTA ATA CGA CTC ACT ATA GCC CGG ATA GCC TAG TC-3’ and the reverse primer 5’-GGG GCT GCA GTG GTG CCC GGA GCG TGA-3’. The PCR product was inserted into the region between the EcoR I and Pst I sites. An intron region in MAt4696 was removed by iPCR with 5’-ATC TGG AGG TCG CGT GTT CG-3’ and 5’TAT GAG TCT GGC GCC CCA AC-3’. The PCR product was circularized by treating with T4 polynucleotide kinase and T4 DNA ligase. In vitro run off transcription was performed following an established method [26]. The tRNAMet transcript was purified by HiTrap Q HP column (5 ml) chromatography with a linear gradient (100 ml) from 0.4 to 0.8 M NaCl. The tRNAMet transcript was recovered from the fractions by ethanol precipitation. Guanine exchange assay The assay was performed essentially using a previously described method [9]. Briefly, the reaction was performed at 37 °C in 100 mM HEPES–NaOH (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT and 43.2 lM [14C]-guanine hydrochloride, with various concentrations of ArcTGT and tRNAMet transcript. Aliquot was spotted onto Whatman 3MM filter paper and the reaction product on the filter was precipitated in 5% trichloroacetic acid (TCA). The radioactivity retained on filters after several washings in 5% TCA was measured with a liquid scintillation counter, AccuFLEX-LSC 7200 (Hitachi Aloka Medical, Ltd., Mitaka, Japan). LC–MS analysis PreQ0 exchange reaction (50 ll) was carried out in 100 mM HEPES–NaOH (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, 16 lM tRNAMet transcript, 1 mM preQ0 and either 2 lg recombinant ArcTGT or no enzyme at 37 °C for 1 h, and then the reaction product was recovered by phenol/chloroform extraction and ethanol precipitation. The precipitate was resuspended in 100 ll of ultrapure water and the solution was analyzed using an XevoTM QTof mass spectrometry instrument (Waters, Milford, USA). Liquid chromatography analysis was performed using an ACQUITY UPLCÒ BEH C18 1.7 lm 2.1  100 mm Column (Waters) with a linear gradient from 100 mM 1,1,1,3,3,3-Hexafluoro-2-propanol (HIFP), 8.6 mM Triethylamine (TEA) to 50 mM HIFP, 4.3 mM TEA, 50% methanol. Results Purification of a split-type ArcTGT from M. acetivorans Initially, we aimed to purify a native split-type ArcTGT from M. acetivorans cell extract. Here, one unit of the enzyme activity was defined as the amount catalyzing the incorporation of 1 pmol of guanine into M. acetivorans tRNAMet transcript per min. Table 1 shows the yield and specific activity of the enzyme preparation at each step. The yield from the final step using the Mono Q column was low, at 0.19% and was predominantly attributed to the low recovery when the HiTrap Q HP column was used at the fifth step. Although a variety of commonly available columns were tested at the fifth step, we could not identify a column that was superior to the HiTrap Q HP column. Similarly, it was difficult to choose an appropriate column for the third and fourth step. Consequently, the use of Phenyl HP and gel filtration columns led to the best overall yield result among the various columns tested (data not shown). We could, however, achieve high-yield recovery during the final

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Table 1 Purification of the native ArcTGT from M. acetivorans. Step

Protein (mg)

Activity (U)

Specific activity (U/mg)

Purity (fold)

Yield (%)

S100 Ammonium sulfate Phenyl HP Sephacryl S-200 Q HP Heparin HP Mono Q

3100 1600 47 24 0.048 0.014 0.002

29,000 27,000 7600 4800 100 59 54

9.4 17 160 200 2105 4370 27,000

1.0 1.9 17 21 220 470 2900

100 94 26 17 0.34 0.20 0.19

vidually overexpress both MA4419 and MA0121 as C-terminally His6-tagged proteins. However, MA4419 was highly insoluble in cells and MA0121 was not expressed at all (data not shown). Therefore, we decided to co-express MA0121 and C-terminally His6-tagged MA4419. As a result, both proteins were effectively overexpressed (Fig. 3, lane 1). Because MA4419 was a C-terminally His6-tagged protein, we used an Ni-NTA column as the first step of the purification procedure. We detected only two proteins, which corresponded to MA4419 and MA0121, from the result of SDS PAGE at the final step (Fig. 3, lane 5). In the final step, the specific activity of the recombinant ArcTGT (31,000 U/mg) resulted in similar values to that of the native ArcTGT (27,000 U/mg) (Table 1 and 2). This result shows that the recombinant ArcTGT fused to a His6 tag has almost the same specific activity as the native ArcTGT.

Characterization of M. acetivorans ArcTGT The molecular weight of the split ArcTGT complex was investigated using a Superdex 200 gel filtration column (Fig. 4). The recombinant ArcTGT was mainly eluted at fraction number 14 and its molecular weight was estimated as approximately 150 kDa from the elution pattern of the marker proteins. Because the split ArcTGT complex contains MA0121 (19 kDa) and MA4419 (54 kDa) at an equal molar ratio, the split ArcTGT is supposed to form a heterotetrameric structure. Moreover, because the activity of native ArcTGT also eluted in the same fraction as the recombinant ArcTGT (data not shown), native ArcTGT is also thought to form the a2b2-type heterotetrameric structure. A survey of the optimal conditions for M. acetivorans ArcTGT activity with respect to pH and Mg2+ concentration revealed that the maximum activity was achieved at pH 7.5 in 10 mM MgCl2 Fig. 2. Isolation of native ArcTGT from M. acetivorans. (A) Native ArcTGT was finally isolated on a Mono Q column. Reaction mixtures (10 ll) containing 5 ll each of the indicated column fractions were incubated at 37 °C for 15 min. Aliquots (10 ll) were withdrawn and the radioactivity was quantified. DPM means disintegration per minute. (B) Aliquots (20 ll) of the indicated fractions were analyzed on SDS– PAGE and stained with Coomassie brilliant blue. The positions and sizes of marker proteins are indicated on the left.

two steps. Fig. 2 shows purified fractions collected from a Mono Q column during the final step. Only two proteins were detected in the final active fractions, with an apparently equal molar ratio. The proteins were identified as MA0121 and MA4419, respectively, by mass spectroscopy (data not shown). Based on its genome information, M. acetivorans ArcTGT is predicted to consist of MA4419 and MA0121, corresponding to the N-terminal and C-terminal domains of the full-size type, respectively, and the molecular weight of each subunit is 55 kDa and 19 kDa. Thus, these results indicate that the split-type ArcTGT forms a tight complex in the cell. Overexpression and purification of a recombinant ArcTGT We attempted to express a recombinant ArcTGT in E. coli cells and to purify it from the cell extract. Initially, we planned to indi-

Fig. 3. SDS–PAGE analysis of recombinant ArcTGT purification. Lane 1, S30; lane 2, protein eluted from Ni-NTA column; lane 3, protein after a Superdex 200 gel filtration column; lane 4, protein after a Q HP column; lane 5, protein after a Heparin HP column. M indicates molecular weight markers (kDa).

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Y. Nomura et al. / Protein Expression and Purification 88 (2013) 13–19 Table 2 Purification of the recombinant ArcTGT from E. coli culture. Step

Protein (mg)

Activity (U)

Specific activity (U/mg)

Purity (fold)

Yield (%)

S30 Ni-NTA superflow Superdex 200 Q HP Heparin HP

12 1.1 0.4 0.14 0.042

28,000 14,000 5800 3000 1300

2300 12,000 15,000 21,000 31,000

1.0 5.4 6.5 9.5 14

100 48 21 11 4.7

Fig. 4. The profile of the split ArcTGT structure. Recombinant ArcTGT was analyzed by a gel filtration column, Superdex 200. (A) The elution profiles were measured by UV absorbance. A280 is plotted against each fraction. The column was calibrated with marker proteins, glutamate dehydrogenase (290 kDa), aldolase (158 kDa), lactate dehydrogenase (140 kDa), and enolase (67 kDa), indicated by arrows. (B) Aliquots (10 ll) of the indicated column fractions were analyzed on SDS–PAGE and stained with Coomassie brilliant blue. M indicates molecular weight markers (kDa).

(Fig. 5). In this study, all assays were performed under these optimal conditions. The kinetic parameters of the native and recombinant ArcTGTs were then compared (Table 3). Although the kinetic parameters of native ArcTGT could be measured only once because of the small amount of native ArcTGT purified, the Kcat/Km values obtained for the native and recombinant ArcTGT were approximately identical. This result suggests that recombinant ArcTGT can be used in various reactions in place of native ArcTGT. Preparation of preQ0-tRNA with recombinant M. acetivorans ArcTGT Using LC–MS analysis, we investigated whether the recombinant ArcTGT could actually produce preQ0-tRNA (Fig. 6). A major peak consistent with the molecular mass of G15-tRNA (24,885 Da) was observed in the absence of ArcTGT and a proximal peak (24,906 Da) corresponded to sodium adducts. In contrast, a

Fig. 5. The profiles of pH (A) and metal (B) for ArcTGT. (A) Reaction mixtures (20 ll) containing 100 mM either MES–NaOH (pH 5.8, 6.0, 6.5) (circles), HEPES–NaOH (pH 7.0 and 7.5) (diamonds), or TAPS–NaOH (pH 8.1, 8.5 and 9.0) (squares), 11 lM tRNAMet and 0.4 lg recombinant ArcTGT were incubated at 37 °C for 5 min. Aliquots (10 ll) were withdrawn and the radioactivity was quantified. (B) Reaction mixtures (20 ll) containing increasing concentrations of MgCl2 (0, 5, 10, 15 and 20 mM), 11 lM tRNAMet and 0.4 lg recombinant ArcTGT were incubated at 37 °C for 5 min. Aliquots (10 ll) were withdrawn and the radioactivity was quantified. Each assay was carried out three times independently. DPM means disintegration per minute.

Table 3 The kinetic parameters of ArcTGT, based on tRNAMet as the substrate. 3

ArcTGT

Kcat (10

s

Recombinant Native

66.7 ± 5.05 31.1

1

)

Km (lM)

Kcat/Km (M

2.1 ± 0.34 1.2

3.1  104 2.7  104

1

s

1

)

Reaction mixtures containing increasing concentrations of tRNAMet (from 0.5 to 11 lM) and either 0.29 lM recombinant or 10 nM native ArcTGT were incubated at 37 °C.

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Fig. 6. LC–MS analysis of preQ0-tRNA generated by recombinant ArcTGT. Reaction mixtures (50 ll) containing 16 lM tRNAMet and 1 mM preQ0 and either no enzyme (A) or 2 lg recombinant ArcTGT (B) were incubated at 37 °C for 1 h.

peak consistent with that of preQ0-tRNA (24,909 Da) was detected when ArcTGT and preQ0 were added to the reaction, with a concomitant decrease in the G15-tRNA peak. A proximal peak (24,930 Da) corresponding to the sodium adducts was also observed.

Discussion We believe that it is important to compare native and recombinant proteins in various ways. Currently, it is difficult to cultivate archaeal cells and purify target proteins in large quantities because archaeal species usually grow under extreme environmental conditions. Therefore, the investigations of proteins originating from Archaea have so far used recombinant proteins produced in E. coli cells, with the assistance of genomic information accumulated in databases and genetic engineering. As a result, there has been little experimental evidence gathered for archaeal proteins on whether or not the characteristics of a recombinant protein are virtually identical to those of the native protein. In this study, we succeeded in demonstrating the validity of recombinant ArcTGT. As an exception, we found different behaviors between native and recombinant ArcTGT during protein purification. Based on the primary sequence of ArcTGT, the theoretical isoelectric point is calculated as pH 7.6 but the native and recombinant ArcTGTs were

not effectively purified using a cation exchange column, S HP, at pH 7.0. Alternatively, an anion-exchange column, Q HP, could be effectively used for the purification of the recombinant ArcTGT. However, when Q HP was used to purify the native ArcTGT, it was eluted in a broad peak, compared with the elution profile of the recombinant ArcTGT. Probably, the native ArcTGT is associated with other proteins or RNA in the crude preparations. Therefore, the recovery of the native ArcTGT from the Q HP column was low. The native and recombinant ArcTGTs exhibited similar results to one another in the other analyses. Comparing M. acetivorans ArcTGTs with other TGTs, M. acetivorans ArcTGTs showed higher Km values for the tRNA substrate, 2.1 ± 0.34 lM (recombinant) and 1.2 lM (native) respectively (Table 3), than that of P. horikoshii, E. coli and human TGT (0.57, 0.19 ± 0.05 and 0.34 ± 0.04 lM respectively) [20,27,28]. The decrease in affinity may be attributed to the split type. However, there have been few studies on split ArcTGT, so further investigations are required to reveal the differences between the full-size and split types. It has been demonstrated that the full-size ArcTGT forms a homodimeric structure (a2) [16]. By analogy with the quaternary structure of the full-size ArcTGT, split ArcTGT has been assumed to form a heterotetrameric structure (a2b2), but there has been no definitive evidence for this. In this study, for the first time, we clearly showed native ArcTGT was isolated as a heterotetrameric structure. Therefore, it is strongly suggested that the a and b subunits tightly interact with each other within the cell and that no other protein, for example ArcS (responsible for archaeosine synthesis), forms a complex with ArcTGT. Herein, we successfully prepared a recombinant ArcTGT with the same activity as native ArcTGT. This was obtained by co-overexpression of the two split subunits, while expression of one subunit alone resulted in no recovery of the proteins in the soluble fractions. These results imply that the interaction between the Nterminal and C-terminal domains may contribute to the conformational stability of ArcTGT. This recombinant ArcTGT is expected to be useful for further experimental research, because preQ0-tRNA, which can be used as a substrate in assays to detect ArcS activity, can be effectively produced under the optimal conditions we determined. Recently, several candidate proteins involved in archaeosine synthesis have been reported [14,15]. However, its catalytic mechanism and structural information currently remain ambiguous. We are planning to purify the enzymes responsible for archaeosine synthesis from M. acetivorans cell extract using the preQ0tRNA prepared herein. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T.Y.). References [1] S. Nishimura, Structure, biosynthesis, and function of queuosine in transfer RNA, Prog. Nucl. Acid Res. Mol. Biol. 28 (1983) 49–73. [2] D. Iwata-Reuyl, Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA, Bioorg. Chem. 31 (2003) 24–43. [3] F. Harada, S. Nishimura, Possible anticodon sequences of tRNAHis, tRNAAsn, and tRNAAsp from Escherichia coli B. Universal presence of nucleoside Q in the first position of the anticodons of these transfer ribonucleic acids, Biochemistry 11 (1972) 301–308. [4] F. Meier, B. Suter, H. Grosjean, G. Keith, E. Kubli, Queuosine modification of the wobble base in tRNAHis influences ‘in vivo’ decoding properties, EMBO J. 4 (1985) 823–827. [5] R.C. Morris, K.G. Brown, M.S. Elliott, The effect of queuosine on tRNA structure and function, J. Biomol. Struct. Dyn. 16 (1999) 754–774. [6] J.M. Gregson, P.F. Crain, C.G. Edmonds, R. Gupta, T. Hashizume, D.W. Phillipson, J.A. McCloskey, Structure of the archaeal transfer RNA nucleoside G+-15 (2amino-4, 7-dihydro-4-oxo-7-b-D-ribofuranosyl-1H-pyrrolo[2,3-d]pyrimidine5-carboximidamide (Archaeosine)), J. Biol. Chem. 268 (1993) 10076–10086.

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