Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA

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 (S(0)) 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 (s(4)U), a modified nucleotide in tRNA. In addition to IscS, s(4)U 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 s(4)U. 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 S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U.     

The rhodanese domain of ThiI is both necessary and sufficient for synthesis of the thiazole moiety of thiamine in Salmonella enterica

In Salmonella enterica, ThiI is a bifunctional enzyme required for the synthesis of both the 4-thiouridine modification in tRNA and the thiazole moiety of thiamine. In 4-thiouridine biosynthesis, ThiI adenylates the tRNA uridine and transfers sulfur from a persulfide formed on the protein. The role of ThiI in thiazole synthesis is not yet well understood. Mutational analysis described here found that ThiI residues required for 4-thiouridine synthesis were not involved in thiazole biosynthesis. The data further showed that the C-terminal rhodanese domain of ThiI was sufficient for thiazole synthesis in vivo. Together, these data support the conclusion that sulfur mobilization in thiazole synthesis is mechanistically distinct from that in 4-thiouridine synthesis and suggest that functional annotation of ThiI in genome sequences should be readdressed. Nutritional studies described here identified an additional cysteine-dependent mechanism for sulfur mobilization to thiazole that did not require ThiI, IscS, SufS, or glutathione. The latter mechanism may provide insights into the chemistry used for sulfur mobilization to thiazole in organisms that do not utilize ThiI.     

Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate

ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S(2-) rather than S(0).     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.     

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

4-Thiouridine (s⁴U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s⁴U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s⁴U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s⁴U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s⁴U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s⁴U. In the absence of an RLD, MMP1354 uses Cys²⁶⁵ and Cys²⁶⁸ located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S²⁻ is a physiologically relevant sulfur donor for s⁴U formation catalyzed by MMP1354 (K_m for Na₂S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s⁴U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.     

The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD

IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s(4)U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s(4)U in its tRNA. Assays of cell-free extracts isolated from the iscS(-) strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s(4)U, the iscS(-) strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating that iscS is required specifically for thiazole biosynthesis. The growth rate of the iscS(-) strain is half that of the parent strain in rich medium. When the iscS(-) strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s(4)U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS(-) strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS(-) strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli, neither can complement loss of iscS when each is overexpressed in the iscS(-) strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides.     

The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form

After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.     

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

Thiamine pyrophosphate is a required coenzyme that contains a mechanistically important sulfur atom. In Salmonella enterica, sulfur is trafficked to both thiamine biosynthesis and 4-thiouridine biosynthesis by the enzyme ThiI using persulfide (R-S-S-H) chemistry. It was previously reported that a thiI mutant strain could grow independent of exogenous thiamine in the presence of cysteine, suggesting there was a second mechanism for sulfur mobilization. Data reported here show that oxidation products of cysteine rescue the growth of a thiI mutant strain by a mechanism that requires the transporter YdjN and the cysteine desulfhydrase CdsH. The data are consistent with a model in which sulfide produced by CdsH reacts with cystine (Cys-S-S-Cys), S-sulfocysteine (Cys-S-SO₃⁻), or another disulfide to form a small-molecule persulfide (R-S-S-H). We suggest that this persulfide replaced ThiI by donating sulfur to the thiamine sulfur carrier protein ThiS. This model describes a potential mechanism used for sulfur trafficking in organisms that lack ThiI but are capable of thiamine biosynthesis.     

Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli

Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur. Cysteine is the intracellular sulfur source for each of these modified bases. We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E. coli. Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps. In addition to iscS, E. coli contains two other nifS homologs, csdA and csdB, each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis. Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content. It was found that unfractionated tRNA isolated from the iscS mutant strain contained <5% of the level of sulfur found in the parent strain. High-pressure liquid chromatography analysis of tRNA nuclease digests from the mutant strain grown in the presence of [(35)S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase. As expected, digests from the iscS mutant strain contained 6-N-dimethylallyl adenosine (i(6)A) in place of 6-N-dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm(5)U) instead of 5-methylaminomethyl-2-thiouridine. Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6-N-dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent. This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains. An overall scheme for thionucleoside biosynthesis in E. coli is discussed.     

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.     

The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA.

The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.     

Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation

The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s(4)U) and 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s(2)C) and N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm(5)s(2)U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms(2)io(6)A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms(2)io(6)A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.     

Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions

The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking.     

半胱氨酸脱硫酶突变体H104Q,E156Q,D180G,Q183E和K206A通过改变对磷酸吡哆醛的结合影响酶活性

大肠杆菌半胱氨酸脱硫酶（cysteine desulfurase,IscS）是一类依赖磷酸吡哆醛（pyridoxal phosphate,PLP）的同质二聚体的酶.IscS能催化游离底物L-半胱氨酸脱硫,生成L-丙氨酸和单质硫.在此催化过程中,可形成与酶结合的半胱氨酸过硫化物中间物,并出现了7种具有不同特征性吸收峰的中间反应物.为了研究PLP的结合及中间反应物的形成及累积,对IscS中与PLP结合相关,及IscS半胱氨酸活性口袋中特定氨基酸残基位点（His104,Glu156,Asp180,Gln183和Lys206）进行定点突变,结果发现：1）IscS突变体H104Q、D180G、Q183E、K206A对PLP的结合能力具有不同程度的减弱,酶的活性明显降低甚至消失,PLP与蛋白结合的特异吸收峰消失,或发生明显偏移并出现新的吸收峰,且这些新出现的吸收峰又与蛋白形成的各种中间反应物的吸收峰一致|2）IscS突变体E156Q的活性增高,PLP与蛋白结合的吸收峰明显增加.这些结果都表明,IscS氨基酸残基可通过影响PLP的结合及质子转移引起催化过程中不同中间反应物的形成及累积,同时提高或降低蛋白的活性.     

Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways

tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s(2)C), 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms(2)io(6)A (10-fold) and s(2)C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms(2)io(6)A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s(4)U and the s(2)U moiety of (c)mnm(5)s(2)U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s(2)C and MiaB in the synthesis of ms(2)io(6)A) in the transfer of sulfur to the tRNA.     

Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.