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J Biol Chem, Vol. 275, Issue 15, 10727-10730, April 14, 2000

ACCELERATED PUBLICATION
Evidence for the Transfer of Sulfane Sulfur from IscS to ThiI during the in Vitro Biosynthesis of 4-Thiouridine in Escherichia coli tRNA^*

Ravi Kambampati and Charles T. Lauhon

From the School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S⁰) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s⁴U), a modified nucleotide in tRNA. In addition to IscS, s⁴U synthesis in E. coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor. We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s⁴U. Treatment of ThiI with 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis. Both ATP and tRNA can protect ThiI from I-AEDANS inhibition. Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E. coli tRNA^Phe. Together, these results suggest that ThiI is a recipient of S⁰ from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s⁴U.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Sulfur trafficking within the cell remains a poorly understood area. The biochemical steps for sulfur incorporation into biotin, lipoic acid, and thiamin, as well as macromolecules such as protein Fe-S clusters and thionucleosides in tRNA, have not been fully elucidated. The biosynthesis of thiamin and 4-thiouridine (s⁴U)¹ in tRNA has been linked by the observation that some mutants lacking s⁴U in their tRNA were also auxotrophic for thiamin (1). In two independent studies, the thiI gene was isolated and found to complement both s⁴U (2) and thiamin (3) deficient mutants in Escherichia coli and Salmonella typhimurium, respectively. We recently reported that IscS, a NifS homolog from E. coli, can mobilize sulfur from cysteine for the in vitro synthesis of s⁴U in E. coli tRNA (4). The formation of s⁴U requires IscS and ThiI as well as Mg-ATP and L-cysteine as the sulfur donor. Using UV spectroscopy and HPLC analysis, we have confirmed that s⁴U is the product of the in vitro reaction (4). All tRNA substrates lacking a uridine in position 8, the natural site of s⁴U modification, are completely inactive in our system, suggesting that the modification is restricted to this position.² We have also found that IscS, among multiple enzymes in E. coli with cysteine desulfurase activity, is specifically involved in sulfur transfer to tRNA in vitro (4).

Early work by Lipsett and co-workers (5) showed that two factors, A and C, mediated s⁴U synthesis. Two genes, nuvA and nuvC, which were later identified in E. coli near UV-resistant mutants, were reported to lack s⁴U and were complemented by Factors A and C, respectively (1, 6). IscS requires PLP for activity analogous to Factor C; however, the location of iscS in the E. coli genome (57.3 min) is different from that reported for nuvC (42-46 min). The thiI gene was previously reported to complement a nuvC mutation (7), however, ThiI does not bind PLP (4, 7). Thus, the assignment of iscS and thiI as nuvA or nuvC remains unclear.

IscS was previously characterized as a cysteine desulfurase that catalyzes the formation of alanine and sulfane sulfur (S⁰) from cysteine (8). During this reaction, a cysteine persulfide is formed in the IscS active site. In the presence of excess DTT or other thiols, the products of the reaction are alanine and sulfide. Flint (8) has proposed a scheme in which IscS functions as a S⁰ donor for Fe-S cluster synthesis. We have found that ThiI stimulates the cysteine desulfurase activity of IscS 2-fold (4). Gel mobility shift studies (reported herein) show that ThiI and not IscS binds to unmodified E. coli tRNA^Phe. We therefore hypothesized that the persulfide sulfur formed at the active site of IscS may be transferred to ThiI for reaction with an activated form of uridine 8 in the tRNA to form s⁴U. To test this hypothesis, we sought to determine the nature of sulfur transfer in the biosynthesis of s⁴U in E. coli tRNA. In this paper, we report evidence that ThiI binds tRNA, accepts sulfur from IscS, and functions as the ultimate sulfur donor for s⁴U formation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

General-- L-[³⁵S]Cysteine and [-³²P]ATP were purchased from NEN Life Science Products. TPCK-treated trypsin, ATP, cysteine, and all other biochemicals were from Sigma. Overproduced and purified ThiI and IscS were prepared as described previously (4). Unmodified tRNA^Phe was prepared by linearization of pCF23 (a gift from Dr. Rachel Green, Johns Hopkins Medical School, Baltimore, MD) with BstNI and runoff transcription essentially as described (9). E. coli tRNA^Phe transcript was labeled with ³²P by substituting ATP with [-³²P]ATP in the in vitro transcription reaction mixture.

Gel Mobility Shift Assay-- ³²P-Labeled tRNA (3.7 µM) was incubated with varying amounts (0.9-7.2 µM) of ThiI or IscS in 20 µl of buffer A (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM Mg(OAc)₂, 1 mM DTT, 1 mM PMSF, 3% glycerol) for 5 min at 37 °C. Samples were then applied to 8% native polyacrylamide gels, and electrophoresis was performed using 90 mM Tris/90 mM boric acid and 2.5 mM EDTA at 4 °C. The gels were then dried using gel drying frames and exposed to x-ray film.

Tryptic Digestion-- ThiI (0.13 mg/ml) in buffer A minus PMSF was digested at 37 °C with TPCK-trypsin at a ThiI/trypsin ratio of 83:1 (w/w). In the protection experiments, ThiI was preincubated with 1 mM ATP or 1 mM cysteine or tRNA (at a molar ratio of 1:13) at 37 °C for 5 min in buffer A minus PMSF, and then reaction mixtures were digested as above. Aliquots were removed at various time intervals and analyzed by SDS-PAGE. Gels were quantitated using a densitometer and ImageQuant software (Molecular Dynamics). The intensity of the bands was quantitatively analyzed by the volume quantitation method, and the object average option was used for background correction.

Analysis of Interaction between ThiI and IscS during s⁴U Synthesis-- IscS (0.9 µM) was incubated with increasing amounts of ThiI (0-1.4 µM) as indicated in the figure legends. Reactions were performed either in the presence or absence of 1 mM ATP in 25 µl of buffer A containing 10 µM [³⁵S]cysteine (4000 cpm/pmol), 20 µM PLP. BSA, carbonic anhydrase, and unlabeled tRNA^Phe transcript were also present in the reactions where indicated. Incubations were for 10 min at 37 °C. An equal volume of SDS sample buffer without 2-mercaptoethanol was added to the assay mixtures, which were then heated to 70 °C for 2-5 min and subjected to electrophoresis on 10% SDS-polyacrylamide gels. Proteins were visualized by Coomassie staining followed by PhosphorImager analysis (Molecular Dynamics). Labeled bands were quantitated as described above.

Inhibition of ThiI Catalyzed s⁴U Formation by I-AEDANS-- ThiI (3.0 µg, 54 pmol) in 30 µl of 50 mM Tris-HCl, pH 8.0, 50 mM KCl was incubated with 500 pmol of I-AEDANS (2.3-fold molar excess of cysteines in ThiI) at 25 °C for 30 min in dark. In the experiments in which the protective effect of substrates was assessed, 1.6 mM ATP, 1.9 mM GTP, 16.7 µM [³⁵S]cysteine, or 5.0 µg of unmodified tRNA^Phe were included in the incubation mixture. Reactions were quenched with DTT (8.2 mM final concentration) and diluted to 50 µl with sulfurtransferase assay components. The standard assay mixture is composed of buffer A containing 1 mM ATP, 10 µM [³⁵S]cysteine (1338 CPM/pmol), 5.0 µg of unmodified tRNA^Phe, 20 µM PLP, and 0.2 µg of IscS. For maximal sulfur mobilization, we used saturating amounts of IscS in the assays. The concentrations of ThiI used in the inhibition studies were within the linear range of the assay. The sulfurtransferase assay was carried out at 37 °C for 30 min, and samples were applied to DEAE-81 filters (2.4 cm, Whatman). The filters were washed once with 0.5 M Tris-HCl, pH 9.0, for 30 min and three times with 0.3 M KCl, 20 mM cysteine for 5 min. The filters were finally washed three times with H₂0 in a Buchner funnel and counted in a liquid scintillation counter.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

tRNA Binding Studies-- We first looked for tRNA binding by each of the protein factors involved in the biosynthesis of s⁴U using a gel mobility shift assay. Fig. 1 shows the effect of increasing concentrations of either ThiI (lanes 2-5) or IscS (lanes 8-11) on the mobility of ³²P-labeled unmodified E. coli tRNA^Phe. Analysis by native PAGE indicates that only ThiI is able to shift the mobility of the tRNA substrate. The addition of an excess of unlabeled tRNA (25-50-fold) could block the observed mobility shift by ThiI (lanes 6-7). To corroborate the gel shift results, we performed protease protection experiments on ThiI (Fig. 2). TPCK-trypsin degrades ThiI into a major fragment with an approximate molecular mass of 45 kDa (designated L in Fig. 2); tRNA protects ThiI from proteolysis (lower left panel). Neither cysteine nor ATP protects ThiI from tryptic degradation at this site. However, ATP lowers the rate of proteolysis of both ThiI and the 45-kDa fragment (Fig. 2, lower right panel). Quantitative analysis of the protein bands in Fig. 2 revealed that ThiI proteolysis was slowed by 2-fold in the presence of ATP. ThiI tryptic digests containing primarily the 45-kDa fragment are devoid of tRNA sulfurtransferase activity (data not shown). Both the gel shift analysis and protease protection experiments clearly show that ThiI binds to tRNA.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Analysis of tRNA interaction with ThiI and IscS. Gel mobility shift was performed using 32P-labeled unmodified tRNAPhe as described under "Materials and Methods." Lane 1, tRNA alone (75 pmol, 68 cpm/pmol); lanes 2-5, tRNA incubated with ThiI in the molar ratios of 1:0.25, 1:0.5, 1:1, and 1:2, respectively; lanes 6-7, 25- and 50-fold excess of unlabeled tRNA added to the incubation mixture in lane 5; lanes 8-11, tRNA incubated with IscS in the molar ratios of 1:0.25, 1:0.5, 1:1, and 1:2, respectively.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Protection of tryptic cleavage of ThiI by unlabeled tRNAPhe transcript. ThiI was digested with trypsin as described under "Materials and Methods." Aliquots (15 µl) containing 2.0 µg of ThiI digest were removed at different time intervals and analyzed by SDS-PAGE. Lane MW, molecular mass markers are indicated on the right. The positions of ThiI and its tryptic peptides, large (L) and small (S), are indicated on the left.

Observation of Transfer of ³⁵S from IscS to ThiI during s⁴U Formation-- We used ³⁵S-labeled cysteine in an attempt to follow the transfer of sulfur during s⁴U synthesis in vitro. Reactions containing an increasing amount of ThiI, with or without ATP and tRNA, were incubated for 10 min and then quenched with SDS buffer and subjected to electrophoresis (Fig. 3). Each lane in Fig. 3 corresponds to a separate reaction mixture. ³⁵S-Labeled bands were visualized by PhosphorImager analysis (Fig. 3, B and C). As expected, only IscS can be labeled with [³⁵S]cysteine alone. In reactions with increasing concentrations of ThiI (Fig. 3B, lanes 3-6), we observed a ³⁵S-labeled band of increasing intensity with mobility identical to ThiI. The proportion of ³⁵S label comigrating with IscS was observed to decrease with an increasing ThiI concentration (Fig. 3B, lanes 4-6). However, ³⁵S label on IscS increased 47% in the presence of small amounts of ThiI (Fig. 3B, lane 3). Interestingly, this increase was not observed in the absence of ATP (Fig. 3C, lane 3).

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Interaction between ThiI and IscS during the synthesis of s4U. ThiI and IscS were incubated with [35S]cysteine and unmodified tRNAPhe. The reactions contained 1 mM DTT, and the incubation mixtures were analyzed by SDS-PAGE. Lane 1, ThiI (1.4 µM); lanes 2-6, IscS (0.9 µM) incubated with 0, 0.18, 0.35, 0.7, and 1.4 µM ThiI, respectively; lane 7, IscS (0.9 µM) incubated with ThiI (1.4 µM) and tRNA (18.8 µM); lanes 8 and 9, IscS (0.9 µM) and BSA (1.8 µM) incubated with 0 and 18.8 µM tRNA, respectively; lane 10, IscS (0.9 µM) incubated with carbonic anhydrase (4.1 µM) and tRNA (18.8 µM). Panel A shows the Coomassie-stained gel. Panels B and C show the PhosphorImager scans of reactions performed in the presence and absence of ATP, respectively. Panels D and E show the quantitation of 35S label relative to IscS in the protein bands assigned to IscS and ThiI, as a function of increasing ThiI concentration. The last column in panels D and E shows the levels of 35S label in the protein bands after the addition of tRNA.

When the tRNA substrate was included in the reaction mixture (Fig. 3B, lane 7), the label corresponding to ThiI and IscS was greatly diminished, and a high intensity band that correlates with the position of the tRNA was observed. Quantitation of the labeled bands shown in Fig. 3B reveals that the amount of label on ThiI and IscS was decreased by 81 and 98%, respectively, in the presence of tRNA (Fig. 3D). Together, these labeling patterns suggest that ThiI is covalently modified with sulfur mobilized by IscS and catalyzes the ultimate sulfur transfer in the synthesis of s⁴U. Under previously reported sulfurtransferase assay conditions (4), ThiI and IscS catalyze the transfer of 0.2 mol of ³⁵S to 1 mol of tRNA. HPLC analysis of ³⁵S-labeled tRNA nuclease digests confirms that s⁴U accounts for >85% of the ³⁵S label. In the present experiment (Fig. 3), the radioactivity observed in ThiI is 1% that of the label on tRNA. The higher intensity of the label in the tRNA results from the greater chemical stability of s⁴U relative to the putative protein persulfides. An additional unidentified labeled band with electrophoretic mobility in between IscS and tRNA was also observed (Fig. 3 B, lane 7). Quantitation of this labeled band gave a value 0.1% of the label in tRNA. Because we do not see this band in the Coomassie-stained gel (Fig. 3A, lane 7), and it is visible by staining with ethidium bromide (not shown), we conclude that it is a nucleic acid contaminant present in the tRNA substrate.

We observed no labeling of the negative control protein BSA (Fig. 3B, lanes 8 and 9), which is known to contain reactive cysteine residues, or of carbonic anhydrase (Fig. 3B, lane 10) when each is substituted for ThiI in the reaction mixture. As expected, IscS, in the absence of ThiI, does not catalyze sulfur transfer to tRNA (Fig. 3B, lanes 9 and 10). In the absence of ATP, the amount of label transferred to tRNA was reduced by more than 98%, and the intensity of the labeled band corresponding to ThiI remained unchanged (Fig. 3C, lane 7, and 3E). As expected, ATP was not required for the transfer of ³⁵S from L-cysteine to IscS and then to ThiI (Fig. 3C, lanes 2-6).

The persulfides were expected to be labile to reducing agents, and therefore we removed mercaptoethanol from the electrophoresis sample buffer. However, the gel samples received 0.5 mM DTT from the reaction mixtures, which is required for ThiI activity. It is perhaps unexpected that we observed ³⁵S-labeled ThiI and IscS in the gel under denaturing conditions. Because the DTT from the reaction mixture is rapidly diluted as the proteins enter the stacking gel, it is possible that a fraction of the persulfides survives long enough to be separated in the gel matrix. Flint (8) has found that the reduction of the IscS persulfide in the absence of DTT is slow, despite the presence of 2.5 mM cysteine as substrate. Attempts to exclude DTT completely in our experiments resulted in the appearance of additional high molecular weight protein bands in the gel, presumably because of oligomerization or aggregation. We also found that the presence of 5 mM DTT in the reactions abolished label on either IscS or ThiI in our gel analysis. Thus, the ³⁵S-label is sensitive to higher DTT concentrations, which is consistent with the proposed persulfide functionality. We removed [³⁵S]cysteine from the reaction mixture after incubation using size exclusion chromatography and analyzed the separated large molecule fraction by SDS-PAGE followed by PhosphorImager analysis. This shows that the separated protein fraction contains ³⁵S-labeled ThiI, and the addition of this fraction to tRNA in the presence of Mg-ATP results in the transfer of label to tRNA (data not shown). This result suggests that ³⁵S-modified ThiI lies on the direct pathway to s⁴U formation in tRNA.

Inhibition of ThiI Catalyzed Formation of s⁴U by I-AEDANS-- The reaction of ThiI with the fluorescent thiol-specific alkylating agent I-AEDANS leads to irreversible inactivation of the enzyme. Fig. 4 shows that ThiI can be protected from I-AEDANS inhibition by adding ATP and, to a lesser extent, tRNA into the reaction mixture. As expected, GTP, which is inactive as a substrate for s⁴U formation, does not protect ThiI from I-AEDANS inhibition. Because tRNA and ATP have a protective effect, we are confident that inactivation of IscS, which is present in saturating amounts, is not the cause for the observed inhibition. Exposure of SDS gels to UV light after reaction with I-AEDANS shows a fluorescent band comigrating only with ThiI. An alignment of amino acid sequences of ThiI from E. coli and homologs from a range of other organisms shows that Cys³⁴⁴ is present in all of the analyzed sequences (data not shown). These results support the possibility that ThiI possesses a reactive cysteine that can accept S⁰ for the synthesis of s⁴U. We are currently attempting to identify the modified cysteine residue(s), as well as prepare site-directed mutants for analysis both in vitro and in vivo.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Protective effects of ATP and tRNA on ThiI inactivation by I-AEDANS. ThiI was incubated with I-AEDANS in the presence of various ligands as described under "Materials and Methods" and assayed for sulfurtransferase activity. 35S incorporation into tRNA is expressed as a percentage of untreated ThiI.

General Scheme for Sulfur Trafficking in the Synthesis of s⁴U-- Based on the model proposed by Flint (8) and the evidence presented in our current work, we propose a scheme for sulfur mobilization and transfer in the biosynthesis of s⁴U in E. coli (Fig. 5). The initial mobilization of sulfur from cysteine results in the formation of S⁰ covalently bound to a cysteine in the active site of IscS as a persulfide (IscS-SSH). This persulfide sulfur is then transferred, presumably to a cysteine residue on ThiI, to give the ThiI persulfide (ThiI-SSH). Finally, in the presence of ATP, the persulfide sulfur from ThiI-SSH is mobilized and transferred to tRNA. The exact mechanism of the mobilization in vivo is not yet clear, but it could involve oxidation of the protein via internal reduction of the persulfide by a cysteine residue to give a disulfide that is reduced later. Alternatively, an external reducing agent could be required to directly mobilize the sulfur from the persulfide. Because native ThiI has not yet been isolated, it is not possible to ascertain the oxidation state of the naturally isolated enzyme. It is interesting to note that in Flint's (8) original isolation of IscS in the absence of reducing agents, the enzyme was found to be part of a mixed disulfide with the phosphopantetheine moiety of acyl carrier protein.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Proposed scheme for the mobilization and transfer of sulfur during s4U biosynthesis in E. coli.

The role of ATP in s⁴U synthesis has not yet been elucidated. Lipsett and colleagues (5) proposed that Factor A utilized Mg-ATP to synthesize a tRNA-derived unstable activated intermediate, which is then converted to s⁴U by Factor C in the presence of cysteine and PLP. Based on sequence homology searches with putative ThiI homologs from various organisms, we have proposed that ThiI uses Mg-ATP to activate the oxygen at the 4-position of uridine for attack by a sulfur nucleophile (4). Our observation that ATP protects ThiI from inhibition by I-AEDANS and lowers the rate of ThiI trypsinolysis is consistent with this suggestion.

The observed in vitro shuttling of S⁰ from IscS to ThiI may represent a general strategy in vivo for efficient sulfur transfer in a number of metabolic pathways (10). It is possible that IscS or other NifS homologs in E. coli transfer S⁰ to a variety of other acceptors, including Fe-S cluster-containing enzymes and other sulfurtransferases. We have recently found that E. coli iscS mutants are viable but are unable to synthesize s⁴U or thiamin.³ It is thus likely that IscS initiates sulfur mobilization for the synthesis of both s⁴U and thiazole in vivo. Current efforts are focused on a full characterization of the iscS phenotype in E. coli and further elucidation of the scope of IscS-initiated sulfur transfer in thionucleotide biosynthesis.

Addendum-- While this paper was in review, a paper was published by Mueller and Palenchar (11) showing that ThiI contains a unique P-loop motif (¹⁸⁵SGGXDS¹⁹⁰) that is common to the pyrophosphate synthetase family of enzymes that catalyze the adenylation and substitution of carbonyl oxygens. We note that ThiI contains significant ATPase activity independent of tRNA,³ as was earlier reported for Factor A (5). Thus far, we have only found evidence of ADP formation. However, it is possible that the tRNA-independent ATP hydrolysis is obscuring our ability to observe AMP formation.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM57002 (to C. T. L.) and by a grant from the University of Wisconsin Graduate School.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: School of Pharmacy, University of Wisconsin, 425 N. Charter St., Madison, WI 53706. Tel.: 608-262-3083; Fax: 608-262-3397; E-mail: clauhon@facstaff.wisc.edu.

² C. T. Lauhon and R. Kambampati, manuscript in preparation.

³ C. T. Lauhon and R. Kambampati, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: s⁴U, 4-thiouridine; I-AEDANS, 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid; S⁰, sulfane sulfur; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; PLP, pyridoxal phosphate; DTT, dithiothreitol; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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