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Originally published In Press as doi:10.1074/jbc.M408562200 on September 2, 2004 Originally published In Press as doi:10.1074/jbc.M408562200 on August 30, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47555-47563, November 12, 2004
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MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA*

Fabien Pierrel{ddagger}, Thierry Douki§, Marc Fontecave{ddagger}, and Mohamed Atta{ddagger}||

From the {ddagger}Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DRDC-CB, Unité Mixte de Recherche 5047 Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier and the §Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Département de Recherche Fondamentale sur la Matière Condensée, CEA-Grenoble, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France

Received for publication, July 28, 2004 , and in revised form, August 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The last biosynthetic step for 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyl-adenosine (i6A) precursor. In this work we have reconstituted in vitro the conversion of i6A to ms2i6A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i6A-containing RNA corresponding to the anticodon stem loop of tRNAPhe is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i6A. In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In all organisms, the nucleosides of transfer RNA (tRNA) undergo different post-transcriptional modifications that are important for their biological activity (1). These modifications are part of the complex process of tRNA maturation and are introduced through the action of many different enzymes on the polynucleotide transcripts (2). Although modified bases are found at different positions in tRNA, the anticodon loop is the most heavily modified region of the molecule and contains the greatest variety of modified nucleosides. To date, a total of 86 structurally distinguishable modified nucleosides in tRNA from many diverse organisms of the three major phylogenetic domains of life have been identified (medstat.med.utah.edu/RNAmods/). Among these modified nucleosides, the so-called thionucleosides containing a sulfur atom have been the subject of particular attention in the recent years.

In Escherichia coli four different thiolated nucleosides have been characterized; these are 4-thiouridine (s4U),1 2-thiocytidine (s2C), 5-methylaminomethyl-2-thiouridine (mnm5s2U), and 2-methylthio-N6-isopentenyl-adenosine (ms2i6A). If the synthesis of thiopyrimidines involves a non-redox substitution reaction of an oxygen atom by sulfur, the reaction leading to the synthesis of 2-methylthio-N6-isopentenyl-adenosine (ms2i6A) consists of a chemically highly challenging aromatic C-H to C-S bond conversion. This is an oxidation reaction whose mechanism has yet to be investigated. The modified nucleoside ms2i6A is found at position 37, next to the anticodon on the 3'-position in almost all eukaryotic and bacterial tRNAs that read codons beginning with U except tRNAI,V Ser (3).

Initial studies on ms2i6A biosynthesis in E. coli and Salmonella typhimurium established a requirement for at least two enzymes (4). The first step consists in the addition of the isopentenyl group to the N6 nitrogen of adenosine. This reaction is catalyzed by the well characterized tRNA-isopentenylpyrophosphate transferase enzyme, encoded by the miaA gene (5, 6). The second step, which has been shown by in vivo studies to be dependent on iron, cysteine, and S-adenosylmethionine (AdoMet), corresponds to the sulfur insertion and the methylation at position 2 of the base moiety (7–9). Recently MiaB was shown to be involved at least in the thiolation step on the basis that tRNA from mutant strains lacking a functional miaB gene contain only i6A-37, the product of MiaA activity (4). Concerning the methylation of the sulfur atom, it was postulated that it could require an additional gene named miaC, which has never been identified.

Nothing is known about the mechanism by which MiaB catalyzes the reaction. However, the presence of a strictly conserved canonical cysteine triad Cys-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Cys (Xaa, any amino acid) in the sequence has led to the identification of MiaB as a member of the recently characterized "Radical-AdoMet" family (10) such as the anaerobic ribo-nucleotide reductase-activating enzyme (ARR-AE), pyruvate formate lyase-AE (PFL-AE), lysine aminomutase (LAM), biotin synthase (BioB), and lipoate synthase (LipA) (11–15). In the case of BioB enzyme, which also catalyzes C-H to C-S bond conversion reactions, this motif was shown to provide cysteine ligands for a catalytically essential [4Fe-4S]2+/1+ cluster (16). Such a cluster is present also in MiaB protein (17, 18), and it is thus very likely that MiaB and BioB employ similar radical mechanisms for activation of sulfur and its insertion into their respective substrates.

Recently, we cloned, expressed, and characterized by biochemical and spectroscopic methods the MiaB protein from Thermotoga maritima called MiaBTm (18). The protein is able to assemble an O2-sensitive [4Fe-4S] cluster, which can enjoy both 2+ (oxidized) and 1+ (reduced) redox states. We also showed that a plasmid encoding MiaBTm was able to restore, in vivo, the ms2i6A biosynthesis in an E. coli miaB– strain. Here we report for the first time an in vitro assay for MiaBTm-dependent introduction of a methylthio group and formation of ms2i6A into an i6A-tRNA substrate. Labeling experiments show that AdoMet is the source of the methyl group and evidence is also presented for reductive cleavage of AdoMet into 5'-deoxyadenosine (AdoH). MiaB is thus a complex bifunctional enzyme responsible for both sulfur and methyl insertion, and it uses AdoMet both for substrate radical activation and as a methyl donor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains—E. coli DH5{alpha} was used for routine DNA manipulations. E. coli BL21CodonPlus(DE3)-RILTM (Stratagene), which contains extra copies of genes encoding tRNA with codons rarely used in E. coli (argU, ileY, leuW tRNA genes) was used to produce the recombinant protein MiaBTm. E. coli strain TX3346 miaB- was a gift of Professor Malcolm E. Winkler, University of Texas, Houston. The pTrc99-B plasmid was a gift of Dr. G. Eriani, Strasbourg.

General Procedures—All DNA manipulations were as described previously (19). Enzymes, oligonucleotides, and culture media were purchased from Invitrogen Cergy-Pontoise, France. T4 DNA ligase was from MBI Fermentas Inc. Bacterial alkaline phosphatase and plasmid DNA purification kit, FlexiprepTM, were from Amersham Biosciences. DNA fragments were extracted from agarose gel and purified with High Pure PCR Product Purification kit (Roche Applied Science), DNA sequencing was performed by Genome Express company (Grenoble, France).

Expression and Purification Methods—The MiaBTm protein was overexpressed in E. coli BL21CodonPlus(DE3)-RILTM and purified as previously described, apo and reconstituted forms were obtained as previously described (18). IscS and SufS have been purified as previously described and have been provided by Dr. S. Ollagnier-de-Choudens (20, 21). Flavodoxin, and flavodoxin reductase were provided by Dr. Etienne Mulliez (22). Phenylalanyl tRNA synthetase was overexpressed and purified as described in Ref. 23.

Analytical Methods—Protein concentration was measured by the method of Bradford using bovine serum albumin as a standard (24) and also checked using the bicinchonic acid protein kit assay (Sigma) with the same standard. Iron was determined by the method of Fish (25), and inorganic sulfide was quantified as described by Beinert (26).

Construction of the Synthetic Gene Coding for E. coli tRNAPhe—We constructed a 100-bp long fragment corresponding to mature E. coli tRNAPhe from three complementary pairs of synthetic oligonucleotides. The sequence of these oligonucleotides is: Phe1: 5'-AATTCGCCCGTTGCCCGGATAGCTCAGTCGG-3'; Phe2: 5'-TAGAGCAGGGGATTGAAAATCCCCGTGTCCTT-3'; Phe3: 5'-GGTTCGATTCCGAGTCCGGGCACCAAATTACGCGGGG-3'; Phe4: 5'-GATCCCCCGCGTAATTTGGTGCCCGG-3'; Phe5: 5'-ACTCGGAATCGAACCAAGGACACGGGGATTT-3'; Phe6: 5'-TCAATCCCCTGCTCTACCGACTGAGCTATCCGGGCAACGGGCG-3'.

Internal oligonucleotides Phe2, Phe3, Phe5, and Phe6 (100 pmol each) were phosphorylated in 50 mM Tris-Cl pH 7.6, 10 mM MgCl2, 10 mM DTT, 0.1 mM ATP, with 10 units of T4 polynucleotide kinase for 1 h at 37 °C. A mixture of 25 pmol of each oligonucleotide was heated to 95 °C for 5 min, then cooled slowly to room temperature. Ligation of hybridized oligonucleotides was carried out with 2.5 units of T4 DNA ligase at 18 °C for 15–18 h. Electrophoresis on a 15% non-denaturing TAE-buffered polyacrylamide gel showed that the major ligation product corresponded to that expected for the tRNA gene coding fragment and was also used to purify this fragment. The pTrc99-B plasmid was cleaved by EcoR1 and BamH1 and was combined with the insert. Ligation was carried out under the conditions described above. The ligation mixture was used to transform competent E. coli DH5a. The plasmid DNA of the clone with an insert of the correct size was then sequenced and named pTrc99-B-tRNAPhe.

Overproduction of tRNAPhe and Preparation of tRNAPhe-enriched tRNAs—Overproduction of tRNAPhe was performed using the E. coli TX3346 miaB– strain lacking a functional miaB gene. The transformed cells with pTrc99-B-tRNAPhe were grown overnight in 5 ml of LB medium containing 100 µg of ampicillin/ml. This overnight culture was used to inoculate 5 liters of LB medium. When the culture reached 0.8 OD600, tRNAPhe expression was induced by adding isopropyl-1-thio-{beta}-D-galactopyranoside to a final concentration of 0.5 mM followed by incubation for 15 h at 37 °C. Bacterial cells were harvested by centrifugation, resuspended in 200 mM Tris-Cl pH 8, and then extracted by an equal volume of phenol saturated with 200 mM Tris-Cl, pH 8. After 30 min of vigorous shaking at room temperature, the aqueous phase was collected by low speed centrifugation and extracted again under the same conditions. The small RNAs were precipitated with ethanol, resuspended in 50 ml of 500 mM Tris-Cl, pH 8.8, and incubated at 37 °C for 45 min in order to deacylate the extracted tRNAs. The solution was then neutralized by addition of 10 ml of 1 M sodium acetate, pH 5.1 and RNAs were precipitated with 3 volumes of cold ethanol.

The total RNAs pellet was dissolved in 10 mM Tris-H3PO4, pH 6.3 containing 15% ethanol and 400 mM KCl, and the solution was applied to a Nucleobond column AX10000 (Clontech) equilibrated with the same buffer. The column was then washed extensively with the same buffer and tRNAs were eluted by increasing the concentration of KCl to 650 mM. tRNAs were precipitated with 0.7 volume of cold isopropyl alcohol for 1 h at 4 °C, washed with 70% ethanol, and dried. tRNAs were dissolved in water and stored at –20 °C in 250 mM NaCl and 3 volumes of ethanol. The [14C]Phe acceptor activity of the total tRNAs preparation, obtained from these cells has been assayed by using the E. coli phenylalanyl-tRNA synthetase enzyme (23). The obtained activity was up to 430 pmol per A260 unit to be compared with 90 pmol for total tRNAs isolated from E. coli TX3346 cells not transformed with the overproducing plasmid. Total tRNAs obtained from the tRNAPhe overexpressing E. coli miaB– cells were named i6A-37-tRNAs and were used as a substrate for MiaB protein.

Aminoacylation Assay—The reaction mixture (50 µl) contained 100 mM Hepes, pH 7.5, 30 mM KCl, 10 mM MgCl2, 5 mM reduced glutathione, 2 mM ATP, 100 µg/ml bovine serum albumin, 1 µM phenylalanyl-tRNA synthetase, 50 µM [14C]phenylalanine (25 µCi/µmol), and 20 µg of purified tRNAs. Incubation was 15 min at 37 °C, and the reaction was stopped by the addition of 25 µl of the reaction mixture to a Whatman GF/A filter prewetted with 5% trichloroacetic acid. The filters were washed three times during 5 min in 5% trichloroacetic acid, then two times for 2 min in 95% ethanol, and finally dried at 50 °C during 10 min. The radioactivity was then counted.

Analysis of tRNA Nucleoside Composition by HPLC—i6A-37-tRNAs and the in vitro assay solutions were digested to nucleosides by the method of Gehrke and Kuo (27) by using nuclease P1 and bacterial alkaline phosphatase. 50–100 µg of tRNAs was loaded onto Zorbax SB-C-18 column connected to a HP-1100 HPLC system. The short gradient profile developed by Gehrke and Kuo (27) was used to separate the different nucleosides. The ms2i6A was quantified by converting the area of the corresponding HPLC peak by using the response factor of 808 units of area of the ms2i6A peak detected at 254 nm/nmol of ms2i6A.

In Vitro Enzyme Assay—The assay mixture (20 µl) contained, in 100 mM Tris-Cl pH 7.5, 1 mM AdoMet (or 100 µM [3H3C]AdoMet for labeling experiments), 2 mM dithionite, 150 µM i6A-37-tRNAs, and 70 µM reconstituted MiaB protein. After 90 min of reaction at 37 °C under anaerobic conditions, 80 µl of water was added to the assay mixture, and tRNAs were extracted by phenol treatment and then ethanol-precipitated. tRNAs were digested and analyzed by HPLC for ms2i6A as described above.

Analysis of 5'-Deoxyadenosine (AdoH) by HPLC—The in vitro reaction (30 µl) was stopped by the addition of 3 µl of 1 M HCl in the assay. After 30 min, the volume was completed to 110 µl with water, and the solution was centrifuged 10 min at 14,000 rpm at room temperature. The supernatant was injected onto an HPLC Zorbax SB-C18 column equilibrated with 0.1% trifluoroacetic acid. A linear gradient from 0 to 28% acetonitrile in 0.1% trifluoroacetic acid was performed at 1 ml/min over 15 min. AdoH, detected from its absorption at 260 nm, eluted after 9.5 min. A standard curve established with pure AdoH enabled us to quantify the AdoH produced in our assay directly from the area of the corresponding HPLC peak.

In Vitro Reconstitution of MiaB with Fe/Se—ApoMiaB was obtained as described previously (18). Typically apoMiaB (200 µM) was incubated with 2 mM DTT for 10 min. Then, 2 mM FeCl3, previously reduced anaerobically with DTT, was added to the protein solution, followed by addition of 3 mM selenocystine. Cluster assembly was initiated by addition of 2.5 µM SufS protein, and the Fe-Se cluster incorporation into apoMiaB was monitored by UV-visible light absorption. The solution was then treated with 600 µM EDTA in order to get rid of the excess of iron and finally the reconstituted protein was desalted by chomatography on a Sephadex G-25 column.

Mass Spectrometry Analysis—HPLC-tandem mass spectrometry analyses were performed with a 1100 Agilent chromatographic system coupled with an API 3000 triple quadripolar apparatus (PerkinElmer Life Sciences) equipped with a turbo ionspray electrospray source used in the positive mode. HPLC separation was carried out with a 2 x 150 mm octadecylsilyl silica gel (3-mm particle size) column (Uptisphere, Interchim Montluçon, France) and a gradient of acetonitrile in 5 mM ammonium formate as the mobile phase. The proportion of acetonitrile rose from 0 to 40% over the first 20 min, and the latter composition was maintained for 40 min. The settings of the tandem mass spectrometer were optimized by injection of a pure solution of i6A in order to favor loss of the ribose unit upon collision-induced fragmentation. Mass spectrometry detection was carried out in neutral loss mode in order to obtain a high specificity. In this configuration, pseudo-molecular ions ([M+H]+) and fragments ([M-132+H]+) obtained by collision in the second quadrupole (collision cell) were analyzed in the first and third quadrupoles, respectively. Using this approach, only nucleosides losing their ribose unit were detected. The pseudo-molecular ion of the latter compounds was monitored in a 300–450 mass range.

Light Absorption Spectroscopy—UV-visible absorption spectra were recorded with a Cary 1 Bio (Varian) spectrophotometer connected to the anaerobic glove box by optical fibers.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activity of MiaB Protein in the Conversion of i6Atoms2i6A— Our previous work had shown that complementation of the E. coli TX3346 miaB– strain with the miaB gene (TM0653) from T. maritima restored the production of ms2i6A in these cells in vivo (18). In order to investigate the enzymatic conversion of i6A to ms2i6A in vitro, we established standard reaction conditions for the purified MiaB protein from T. maritima, MiaBTm. Typically the reaction mixture contained MiaBTm, total tRNA enriched with the tRNAPhe substrate, AdoMet, dithionite as a reductant, in a final volume of 20 µl of 100 mM Tris-Cl, pH 8. The reaction was done at 37 °C or 50 °C for 90 min, within an anaerobic glove box. In all standard experiments reported here the purified MiaBTm was the preparation containing a [4Fe-4S] cluster. This protein was obtained after reconstitution of its iron-sulfur center by treatment of apoMiaB under anearobic conditions with an excess of iron and sulfide followed by desalting on Sephadex G-25 as previously described (18). The tRNA substrate was the total tRNAs obtained from the E. coli TX3346 miaB– strain transformed with the expression plasmid pTrc-99B-tRNAPhe for overproduction of tRNAPhe. Thus, this preparation had two characteristics: first, it was enriched with tRNAPhe (about 27% pure based on the acceptor activity value of 1600 pmol of phenylalanine/A260 for pure tRNAPhe, Ref. 28); second, it contained an isopentenyl group at position 6 of adenosine 37 but no methylthio group at position 2 of the same nucleoside. These tRNAs were named i6A-37-tRNAs. After the 90-min incubation, tRNAs were recovered by phenol extraction and ethanol precipitation and then completely hydrolyzed by nuclease P1 and alkaline phosphatase. The resulting hydrolysate was analyzed by HPLC, and the chromatogram presented in Fig. 1A showed the presence of a peak at 57 min (retention time) corresponding to the ms2i6A nucleoside. No ms2i6A could be detected when the MiaB protein, AdoMet, or dithionite was omitted from the reaction mixture. In that case, only the peak at 47 min corresponding to i6A was detected (Fig. 1B). The identity of i6A and ms2i6A was confirmed by their UV-visible spectra and by mass spectrometry (data not shown).



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  		FIG. 1.
In vitro synthesis of ms2i6A. HPLC chromatograms of modified nucleosides in i6A-37-tRNAs (40 µg) after incubation for 90 min at 37 °C with mixA (1 mM AdoMet, 2 mM dithionite, 100 mM Tris-Cl, pH 7.5) in the presence (A) or the absence (B) of 70 µM MiaBTm. After the reaction, the extraction of tRNA and digestion into nucleosides and the subsequent HPLC analysis was as described under "Materials and Methods." The identification of i6A and ms2i6A is based on retention time, UV-visible spectra and was checked by HPLC-tandem mass spectrometry analysis.

 
Production of ms2i6A under these conditions was monitored as a function of reaction time and of MiaB protein concentration. In Fig. 2A it is shown that there is a linear formation of ms2i6A with time to reach a plateau after about 100 min, when the reaction stops for unknown reasons so far. Fig. 2B demonstrates the MiaB dependence of the reaction with the linear formation of ms2i6A as a function of MiaB concentration. These data also show that the reaction is slow and stops after production of about 0.18 ms2i6A per MiaBTm polypeptide. Two other reducing systems have been used in place of dithionite and proved to be functional: light-activated 5-deazaflavin and reduced flavodoxin using flavodoxin reductase and NADPH. However the production of ms2i6A with these reducing systems was about two times lower than with dithionite. These results show for the first time that MiaB is able to catalyze the transformation of i6A to ms2i6A in a tRNA and that this tRNA methythiolation occurs with no need for an additional protein.



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  		FIG. 2.
ms2i6A production as a function of reaction time (A) and quantity of MiaBTm (B). The assay mixture (150 µl), incubated at 37 °C, contained mixA, 70 µM MiaBTm, 250 µg of tRNA-i6A37, and ms2i6A was quantified by HPLC analysis at indicated times (A). Formation of ms2i6A after 100 min incubation in the assay mixture (30 µl) described above containing quantities of MiaBTm ranging from 0 to 2 nmol (B).

 
The Anticodon Stem Loop Is a Substrate for MiaBTm—A seventeen-base RNA oligonucleotide (Scheme 1A) corresponding to the anticodon stem loop of wild-type tRNAPhe was synthesized. This RNA was found to be a substrate of MiaA enzyme, even though it contained only unmodified nucleosides (data not shown), but MiaBTm-dependent formation of ms2i6A absolutely required the presence of the MiaA protein and dimethylallyl diphosphate (DMAPP) in the reaction mixture. In the absence of MiaBTm only i6A could be detected, whereas in the absence of MiaA enzyme or DMAPP neither i6A nor ms2i6A were observed in the HPLC chromatogram. This proves that no methylthiolation can occur in the absence of the isopentenyl group at position 6 of adenosine.



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  		SCHEME 1.
tRNAPhe anticodon stem loop used as a substrate. Conformation of the unmodified oligoribonucleotide (A) and of the i6A containing oligoribonucleotide after modification by MiaA (B). The base pairing is according to Ref. 55.

 
The Origin of the Methyl Group—In the standard assay mixture for in vitro synthesis of ms2i6A the most likely methyl donor is AdoMet. In order to determine whether the MiaB protein displays tRNA-methyltransferase activity the assay was run as described above but in the presence of [3H3C]AdoMet. After 90 min of incubation, tRNAs were recovered and hydrolyzed as described under "Materials and Methods," and the resulting hydrolysate was analyzed by HPLC. Seven fractions were collected (two of them corresponding to the peaks of i6A and ms2i6A), and radioactivity was determined in a scintillation counter. As shown in Fig. 3 the only labeled peak was that corresponding to ms2i6A. No peak and no radioactivity could be detected at the same position of the chromatogram when MiaB protein was omitted from the reaction mixture. We can safely conclude that AdoMet serves as the methyl donor for the synthesis of ms2i6A and that the methyltransferase activity resides in the MiaB protein. Based on this experiment, we established a linear correlation between the area of the HPLC peak of ms2i6A and its radioactivity. With this correlation, we calculated a molar response factor of 808 units of area of the ms2i6A peak detected at 254 nm/nmol of ms2i6A. In all experiments, this response factor was used to quantify the ms2i6A produced.



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  		FIG. 3.
Detection of radioactivity in modified nucleosides of i6A-37-tRNA after reaction with MiaBTm in the presence of labeled 3H3C-AdoMet. The assay mixture and HPLC analysis were as described under "Material and Methods." Fractions 1–7 eluting from the HPLC column were counted for 3H.

 
Reductive Cleavage of AdoMet during the Assay—Radical-AdoMet enzymes proceed by reductive cleavage of AdoMet resulting in formation of methionine and 5'-deoxyadenosyl radical, which is transformed in AdoH after abstraction of an H atom on the substrate (12, 29, 30). Thus formation of AdoH from AdoMet is a signature for this class of enzymes. During the in vitro ms2i6A synthesis assay described above, formation of AdoH was also monitored by HPLC as described under "Materials and Methods." In Fig. 4, curves depicting the ms2i6A (closed circles) and AdoH (open circles) productions are shown as a function of time. The ratio of AdoH with respect to ms2i6A at each time point was constant and equal to 1.5 approximately. This ratio can be rationalized with a model in which only 1 equivalent of AdoMet is required for formation of one molecule of ms2i6A. The observed excess production of AdoH (0.5 equivalent) is likely to be due to an abortive process. No production of AdoH could be detected when i6A-37-tRNAs or MiaB protein were omitted from the reaction mixture. This result strongly suggests that during the conversion of i6A to ms2i6A the MiaB protein uses a 5'-deoxyadenosyl radical, which is produced by reductive cleavage of AdoMet. This supports the hypothesis that MiaB belongs to the Radical-AdoMet family.



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  		FIG. 4.
Time course for the production of ms2i6A and 5'-deoxyadenosine. The reaction mixture (300 µl) incubated at 37 °C contained 1 mg of i6A-37-tRNAs, mixA, 70 µM MiaBTm. At the indicated times, 2 aliquots were taken for ms2i6A and 5'-deoxyadenosine quantification as described under "Materials and Methods."

 
The Source of the Sulfur Atom—The source of the sulfur atom present in thionucleosides has been previously investigated using whole cells (31, 32), cell-free (7), or purified systems (33–35). Cells growing in the presence of [35S]cysteine generate 35S-labeled ms2i6A demonstrating that the primary source of the sulfur atom in ms2i6A is cysteine (36, 37). However, cysteine is certainly not the ultimate sulfur donor, and the question is how sulfur is mobilized from cysteine. Furthermore, in our in vitro assay no cysteine was present and nevertheless production of ms2i6A was obtained. In the in vitro assay described above, the possible sulfur donors are dithinionite (Na2S2O4), AdoMet, and the reconstituted MiaB protein. As a matter of fact, the latter has been treated with sulfide and consequently sulfur atoms may be present not only within the [4Fe-4S] cluster but also in other undefined species (sulfide, persulfide, polysulfides, associated or not with iron). In order to check whether MiaB protein provides the sulfur atom for the synthesis of ms2i6A in vitro, the MiaB protein was labeled with selenium by reconstitution of apoMiaB under anaerobic conditions with an excess of iron and selenide. The selenide was derived from selenocystine by the action of the SufS cysteine desulfurase enzyme. SufS is indeed known for its ability to efficiently catalyze selenium mobilization from selenocysteine (21, 38). The UV-visible spectra of the MiaB proteins reconstituted with iron and sulfide or iron and selenide are shown in Fig. 5. The two spectra are very similar, both having a broad absorption band at 420 nm. Previous reports have indeed shown that [4Fe-4S] and [4Fe-4Se] clusters have very close light absorption properties (39). The iron content of the selenium-substituted MiaB protein was found to vary between 3 and 4 iron atoms per protein, which is comparable to the value found for the MiaB enzyme containing an iron-sulfur cluster.



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  		FIG. 5.
UV-visible absorption spectra of reconstituted [4Fe-4S] or [4Fe-4Se] MiaBTm. The [4Fe-4S] cluster of MiaBTm (dotted line) was reconstituted as described elsewhere (18), and the reconstitution of a [4Fe-4Se] cluster (solid line) was conducted as described under "Materials and Methods." The protein concentrations were 180 µM, and the spectra were registered anaerobically in a 1-mm pathlength curve.

 
The selenium-substituted MiaB protein was used in the standard assay and the nucleosides were analyzed by HPLC. The obtained chromatogram is presented in Fig. 6 (solid line) and shows the presence of a new peak at 59 min and only a tiny peak at 57 min corresponding to ms2i6A nucleoside. On the contrary the chromatogram obtained with [4Fe-4S]-containing MiaB protein displays only one peak at 57 min (Fig. 6, dotted line). Furthermore the UV spectrum of the new product obtained with selenium-substituted MiaB shows small but significant differences with the spectrum of ms2i6A (Fig. 6, inset). The two maxima at 244 and 284 nm, characteristic for the modified nucleoside ms2i6A, are shifted to 251 and 286 nm respectively in the new product. HPLC-MS/MS analyses allowed to show that this new compound was the selenium-substituted modified nucleoside of ms2i6A, a 2-methylseleno-N6-isopentenyl-adenosine (mse2i6A). Indeed, it was readily detected in the neutral loss mode set at –132, strongly suggesting that it was a nucleoside undergoing loss of the ribose unit upon fragmentation. In addition, while the pseudo-molecular ion of ms2i6A was found as expected at m/z = 382 (Fig. 7A), the new compound yielded a major pseudo-molecular ion at m/z = 430 (Fig. 7B). The latter value was consistent with the expected m/z of the pseudo-molecular ion of mse2i6A. This result was further supported, as shown in Fig. 7B, by the observation of a series of pseudo-molecular ions on the mass spectrum of the new compound, in agreement with the distribution of stable isotopes in selenium (Se74 0.9%, Se76 9.0%, Se77 7.6%, Se78 23.5%, Se80 49.8%, Se82 9.2%). All these results demonstrate that the sulfur atom present in ms2i6A does not derive from AdoMet or dithionite but totally from the MiaB protein itself and furthermore that it is introduced in MiaB during treatment with iron and sulfide.



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  		FIG. 6.
HPLC detection of an mse2i6A-modified nucleoside. The chromatograms correspond to the analysis of tRNA-i6A37 modified in vitro by [4Fe-4Se] MiaBTm (solid line) or by [4Fe-4S] MiaBTm (dotted line). The reaction mixture was incubated for 90 min at 37 °C and contained 50 µg of tRNA-i6A37, mixA, 60 µM MiaBTm. Inset, UV-visible spectra of the mse2i6A (solid line) and ms2i6A (dotted line) nucleosides.

 



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  		FIG. 7.
Mass spectra of ms2i6A (A) and mse2i6A (B). The intensity was normalized to 100% on the main peak. The HPLC-MS/MS analyses were performed as described under "Materials and Methods." A, pseudo-molecular ion of ms2i6A was detected at m/z = 382 in hydrolysates of i6A-37-tRNAs modified in vitro by [4Fe-4S] MiaBTm and (B) the pseudo-molecular ion of mse2i6A (main peak at m/z = 430) was detected in hydrolysates of i6A-37-tRNAs modified in vitro by [4Fe-4Se] MiaBTm. The different peaks detected for the pseudo-molecular ion of mse2i6A (m/z at 426, 427, 428 430, and 432) corresponds to the distribution of stable isotopes in selenium (Se76 9%, Se77 7.6%, Se78 23.5%, Se80 49.8%, Se82 9.2%).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There are very few redox reactions of tRNA nucleoside modification. Formation of ms2i6A, which consists in the introduction of a methylthio group at position 2 of adenosine, is one of those rare examples. The MiaB protein, which participates to this reaction is, accordingly, a redox protein and the first and so far unique tRNA-modifying enzyme containing an iron-sulfur cluster required for activity (17, 18). The cluster is a [4Fe-4S] one which shares many spectroscopic and structural properties with those present in members of the Radical-AdoMet enzyme superfamily (12, 18). In these systems, the cluster is chelated by three cysteines belonging to the canonical Cys-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence and the fourth iron has a free coordination site for binding AdoMet (16, 40–43). Until the present investigation, two major questions remained unanswered in the absence of an in vitro enzyme assay for conversion of i6A to ms2i6A. The first one is whether MiaB alone can perform the reaction or an additional, provisionally named MiaC, protein is also required (4). The second one is whether MiaB is a functional Radical-AdoMet enzyme, which uses AdoMet as a source of 5'-deoxyadenosyl radical for substrate activation. The results of the present study, based on the first ms2i6A synthase in vitro assay reported so far, provide an unambiguous answer to both questions.

We first demonstrate that MiaB is able to catalyze the formation of ms2i6A in i6A-37-tRNAs, with no need for an additional protein. Thus the conversion of A into ms2i6A is a process implying two sequential reaction steps and two corresponding enzymes. The first reaction, catalyzed by MiaA, converts A into i6A, whereas the second, catalyzed by MiaB, and not by MiaB and a putative MiaC protein as often speculated (4), converts i6A into ms2i6A. In vitro, the second reaction only requires AdoMet and a source of electrons. The fact that AdoMet is the source of the added methyl group, as shown by labeling experiments (Fig. 3), and not of the sulfur atom implies that MiaB displays two distinct activities: it catalyzes the insertion of a sulfur atom at position 2 of adenosine, possibly with the transient formation of a s2i6A nucleoside, and also a methyltransferase reaction using AdoMet as the methyl donor (Scheme 2).



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  		SCHEME 2.
Proposed mechanism for ms2i6A synthesis. In the first step, one molecule of AdoMet (SAM1) is reductively cleaved by the cluster of MiaB into 5'-deoxyadenosyl radical Ado° (reaction 1). The latter then abstracts the hydrogen atom at the C-2 position of adenosine (reaction 2). The putative transient radical (i6A°) incorporates a sulfur atom from MiaB to yield a postulated s2i6A intermediate (reaction 3). In the final step, methylation by a second molecule of AdoMet (SAM2) completes the formation of ms2i6A (reaction 4). R, ribose.

 
Full modification of tRNAs follows a determined sequence of reactions and in some cases, a given modification of a nucleoside at a given position depends on the presence of specific modifications on the same or on other nucleosides. Here we note that a seventeen-base RNA oligonucleotide corresponding to the anticodon stem loop of the tRNAPhe substrate, with no modified nucleoside except at position 37, which has an i6A moiety (Scheme 1B), is also a substrate of MiaB. This indicates that only the isopentenyl group at position 6 of A-37 and a limited portion of the whole tRNA (the anticodon stem loop) is required for a functional MiaB-tRNA enzyme-substrate complex formation.

This study also confirms that MiaB is a member of the Radical-AdoMet enzyme family on the basis of its ability to catalyze the reductive cleavage of AdoMet. Indeed, we observed, during production of ms2i6A, a parallel formation of AdoH (Fig. 4). We thus propose that the cluster of MiaB has the ability to bind AdoMet, as observed with other members of that class of enzymes, and, in the [4Fe-4S]+ state, to reduce it in order to produce a 5'-deoxyadenosyl radical required for substrate activation (reactions 1 and 2). This proposed mechanism implies two things. First that the production of one molecule of ms2i6A requires the reductive cleavage of only one molecule of AdoMet since there is only one position to activate in the substrate. In fact we observed a slightly larger production of AdoH with regard to ms2i6A (Fig. 4). This behavior has previously been observed with other Radical-AdoMet enzymes (22, 44–46) and might be explained by the great reactivity of the 5'-deoxyadenosyl radical and the imperfect quality of the enzyme-substrate complex under the in vitro conditions, which results in a partial loss of selectivity of the radical. The second implication of the mechanism is that the aromatic hydrogen atom at position 2 of adenine would be directly abstracted by the 5'-deoxyadenosyl radical (Scheme 2, reaction 2). As a matter of fact, in Radical-AdoMet enzymes, the substrate is directly activated by H-atom abstraction by 5'-deoxyadenosyl radical (14, 30). This difficult reaction in the case of MiaB protein is not demonstrated at the present stage and will require further experiments. Finally it is interesting to note that no AdoH formation could be observed in the absence of the tRNA substrate showing a very tight coupling between AdoMet reductive cleavage and substrate radical activation. This has also been observed in the case of lysine aminomutase (47) and pyruvate formate lyase (48).

All these results thus show that MiaB is a bifunctional enzyme catalyzing both sulfur insertion into and methylation of the tRNA substrate and that, for both functions, AdoMet is absolutely required. We thus assume that two molecules of AdoMet are consumed for the production of one molecule of ms2i6A, one by reductive cleavage for production of the 5'deoxyadenosyl radical (Scheme 2, reaction 1) and one in the methyl transfer reaction (Scheme 2, reaction 4). Even though we have no definitive evidence for that we find it likely that MiaB contains two distinct AdoMet binding sites, one in close proximity of the [4Fe-4S] cluster allowing complexation to the accessible iron atom and one for methyl transfer. A potential methyltransferase AdoMet binding domain in MiaB has been proposed on the basis of its amino acid sequence (4).

The last point which deserves a discussion is the nature of the sulfur atom source and the mechanism of sulfur transfer from that source to the activated substrate (Scheme 2, reaction 3), even though we have not specifically addressed this question in the present study. In contrast, during the last years, this question has been extensively addressed in the case of biotin synthase and lipoic acid synthase, two Radical-AdoMet enzymes catalyzing sulfur insertion reactions leading to biotin and lipoic acid synthesis respectively. However, despite much effort, the chemistry involved in these systems is still not understood and is a matter of controversy (14, 15). In starting an investigation of MiaB, a third sulfur insertion system of the same class, we hoped to find solutions to those questions. However, again and as in biotin synthase and lipoic acid synthase, we are stuck with a very slow reaction and a protein, which does not behave as an enzyme since substrate conversion stops much before one turnover has been achieved, even when the reaction is carried out in the presence of large excesses of sulfide. Furthermore, MiaB shares with the two other systems the unique property that the sulfur atom used in the reaction is introduced into the protein itself during reconstitution of the iron-sulfur cluster and can be mobilized to some extent during the reaction to generate ms2i6A. This is nicely demonstrated here with the observation that only selenium was introduced in the modified nucleoside when MiaB was reconstituted with iron and selenide. This experiment also demonstrates for the first time that a [4Fe-4Se] cluster is functional for AdoMet reductive cleavage in a Radical-AdoMet enzyme. Further experiments are required in the case of MiaB to tell something about the nature of the sulfur site present in the protein. At this stage, we leave it opened whether it resides in a second iron-sulfur cluster, distinct from the [4Fe-4S] one involved in AdoMet cleavage, or in a persulfide (or polysulfide) species, two possibilities which have been proposed in the case of biotin synthase (49–54). We should just mention that detailed spectroscopic characterization of MiaBTm did not provide so far any evidence for the presence of an observable cluster distinct from the [4Fe-4S] one (18). There is something crucial in those enzyme systems and common to them that we do not understand and we believe, considering the similarities between them, that once it is solved for one enzyme it will be solved for the others.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence may be addressed: DRDC-CB, CEA-Grenoble, 38054 Grenoble, France. Fax: 0033438789124; E-mail: mfontecave{at}cea.fr. || To whom correspondence may be addressed: DRDC-CB, CEA-Grenoble, 38054 Grenoble, France. Fax: 0033438789124; E-mail: mohamed.atta{at}cea.fr.

1 The abbreviations used are: s4U, 4-thiouridine; s2C, 2-thiocytidine; mnm5s2U, 5-methylaminomethyl-2-thiouridine; i6A-37, the modified nucleoside N6-isopentenyl adenosine at position 37; i6A-37-tRNA, tRNA purified from E. coli miaB– strain overexpressing tRNAPhe; ms2i6A-37, the modified nucleoside 2-methylthio-N6-isopentenyl adenosine at position 37; mse2i6A-37, the modified nucleoside 2-methylseleno-N6-isopentenyl adenosine at position 37; AdoMet, S-adenosylmethionine; Fe-S iron-sulfur cluster; AdoH, 5'-deoxyadenosine; Fe-Se iron-selenium cluster; DTT dithiothreitol; HPLC, high performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank. Dr. G. Eriani (IBMC, Strasbourg) for the gift of the pTrc99-B plasmid and for helping us with the aminoacylation assay. Many thanks to Dr. S. Ollagnier-de-Choudens and Dr. E. Mulliez (DRDC/CB, Grenoble) for providing us with SufS, IscS, flavodoxin, and flavodoxin reductase enzymes and for stimulating discussions.



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 DISCUSSION
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