JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M401261200 on February 21, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19551-19558, May 7, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/19/19551    most recent
M401261200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lauhon, C. T.
Right arrow Articles by Vickery, L. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lauhon, C. T.
Right arrow Articles by Vickery, L. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Substitutions in an Active Site Loop of Escherichia coli IscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesis in Vivo*

Charles T. Lauhon{ddagger}§, Elizabeth Skovran¶||, Hugo D. Urbina**, Diana M. Downs¶, and Larry E. Vickery**

From the {ddagger}School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53705, and **Department of Physiology and Biophysics, University of California, Irvine, California 92697

Received for publication, February 4, 2004 , and in revised form, February 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IscS catalyzes the fragmentation of L-cysteine to L-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys328 resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328 identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cysteine desulfurase IscS is the major cellular catalyst for the mobilization and distribution of sulfur from cysteine for a number of different biosynthetic pathways (Fig. 1) (1–4). It is a member of the NifS family of cysteine desulfurases whose first discovered member, NifS, was characterized as a necessary component for activity of the metalloenzyme nitrogenase (5). It was later shown in both Azotobacter vinelandii and Escherichia coli that the homolog iscS is part of a multicistronic operon (isc) required for the biosynthesis of protein Fe-S clusters (6). IscS is highly conserved among bacteria and eukarya and is required for viability in organisms such as Saccharomyces cerevisiae (7) A. vinelandii (6), and Helicobacter pylori (8). In E. coli (9–11) and Salmonella enterica serovar Typhimurium (12), elimination of iscS results in a viable organism that displays complex nutritional requirements and severely reduced activity of Fe-S cluster enzymes.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1.
Biosynthetic pathways requiring IscS persulfide (boxed) in E. coli. The persulfide transfer role of IscS is schematically illustrated as having two main branches. This includes Fe-S cluster biosynthesis via sulfur transfer to IscU and interaction with other isc cluster proteins (left) and persulfide transfer to tRNA binding proteins for the synthesis of thiouridines mnm5s2U and s4U in tRNA (middle). In addition, a novel role for IscS persulfide has been proposed for sulfur incorporation into the thiazole ring of thiamin (right) (45). E. coli {Delta}iscS mutants are defective in the biosynthesis of all of the pathway metabolites shown, most probably because of defects in the enzymes listed.

 
Most bacteria harbor multiple nifS homologs. E. coli contains iscS and two other paralogs, csdA and sufS (csdB). These enzymes can be placed into two groups, based on sequence of an active site loop that contains an essential cysteine and other criteria (13). By these criteria, E. coli IscS is a group I enzyme, whereas SufS and CSD are members of group II. All use cysteine and selenocysteine as a substrate in vitro with varying efficiency (14). IscS is required for selenium mobilization for the synthesis of the modified nucleoside mnm5se2U1 in tRNA (15), even though SufS utilizes selenocysteine more efficiently in vitro (14). In E. coli, the sufS gene is part of the suf operon that is thought to contribute to Fe-S cluster formation under conditions of cellular stress (16). Deletion of suf genes cause no observable growth defects in E. coli. However, in combination with mutations in iscSUA, all but one suf gene is required for viability (17), suggesting that suf and isc genes have overlapping function.

The mechanism for cysteine desulfuration by NifS was shown by Dean and co-workers to be a novel utilization of pyridoxal phosphate for transferring the sulfur atom of substrate L-cysteine (or selenocysteine) to a cysteine residue in the active site of the enzyme to give a persulfide or hydrodisulfide (18). IscS was isolated based on its ability to provide sulfur for the Fe-S cluster of dihydroxy acid dehydratase and found to have a mechanism similar to NifS (19). Although the exact mechanism of sulfur incorporation in Fe-S clusters remains unclear, several groups have obtained evidence of complexation and persulfide transfer between IscS and IscU (20–22). The amino acid residues in IscS important for interaction with IscU and other potential target proteins (23) have not been defined, although a deletion of the C terminus of IscS reduces sulfur transfer and binding to IscU (21).

A primary role for IscS may be to provide sulfur for Fe-S clusters (24). However, as shown in Fig. 1, a number of cellular processes that do not involve Fe-S enzymes also require iscS. For instance, IscS is required for the biosynthesis of normal levels of all thionucleosides in E. coli (25) and Salmonella typhimurium (26), although two of them are not expected to involve Fe-S cluster enzymes in their synthesis. The biosynthesis of 4-thiouridine (Fig. 2, s4U) involves the transfer of sulfane sulfur from Cys328 of IscS to a cysteine of the ThiI protein (27–29). ThiI, in turn, binds tRNA, activates a specific uridine using Mg-ATP, and transfers the sulfur, during which it is oxidized to a disulfide (30). The synthesis of 2-thiouridine requires IscS in vitro (31) and in vivo (15, 25, 26) and may be similar to s4U synthesis with the MnmA protein as the recipient of the IscS persulfide (31). Both mnmA and iscS mutants contain mnm5U instead of the thiolated derivative, indicating that the modifications at the 2 and 5 positions are formed independently.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2.
Biosynthesis of three naturally occurring tRNA thionucleosides in E. coli. Relevant steps in the biosynthetic pathways for s4U8, mnm5s2U34, and ms2i6A37 are shown as understood. The gene products known to be involved are indicated near the relevant step. The synthesis of a fourth thionucleoside, s2C, is largely uncharacterized but is known to require iscS and ttcA (35).

 
The remaining two thionucleosides, dimethallyl-2-methylthioadenosine (ms2i6A) and 2-thiocytidine (s2C) are both considered to require the action of Fe-S proteins for their synthesis. The miaB gene is required for ms2 incorporation into i6A37 in certain tRNAs (32). MiaB has been shown by sequence homology (33) and in vitro reconstitution (34) to be a 4Fe-4S protein similar in some respects to biotin and lipoate synthases. E. coli mutants lacking either miaB or iscS produce i6A in place of the fully modified base (Fig. 2). Jager et al. (35) report that the ttcA gene required for s2C synthesis contains two required Cys-X-X-Cys motifs (35) that may be indicative of an Fe-S cluster protein. Recently, Leipuviene et al. (36) reported additional evidence consistent with our results that supports the dual role of IscS in thionucleoside biosynthesis as depicted in Fig. 1.

It has been suggested from sequence comparisons and structural data that the active site cysteine in all NifS proteins resides in a loop (37). In the crystal structures of IscS from Thermotoga maritima (37) and E. coli IscS (38), portions of the loop are unstructured. Models of the unresolved region of the loop in E. coli IscS both place Cys328 at least 17 Å from the active site pyridoxal phosphate (38) and support the suggestion that a flexible loop would allow transfer of the persulfide sulfur at a greater distance to protein substrates (37). Because the role of iscS in E. coli requires distribution of sulfur for a variety of pathways, a major question is how specificity is manifested in sulfur transfer from IscS to each of its protein substrates. This study was initiated to probe the active site loop region of E. coli IscS by alanine-scanning mutagenesis. In this work, we report the characterization of two mutants that are defective in Fe-S biosynthesis in vivo but functional in persulfide formation and transfer in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains—The parent strain for the iscS mutant (CL100) used in this study is E. coli MC1061. Preparation and characterization of an inframe iscS deletion in this strain has been described previously (8). E. coli strain OD1190, which has a polar disruption of the iscU gene, was a generous gift of Dr. James Imlay. S. enterica strain DM5420 has been described previously (12) and has a polar MudJ insertion in iscA.

Construction of iscS Alanine Mutants—Alanine mutants were prepared using the QuikChange mutagenesis protocol (Stratagene) and plasmid pCL010, a pET21c-derived plasmid containing wild type iscS sequence (27). Mutant plasmids were isolated, sequenced, and used to transform strain CL100(DE3) (a DE3 lysogen containing a nonpolar iscS deletion) (9). This strain exhibited basal expression of T7 RNA polymerase in the absence of isopropyl-1-thio-{beta}-D-galactopyranoside and hyper-expression in its presence. Mutations of interest were also prepared in the low copy plasmid pCL199, a derivative of pKO3 (39). This plasmid contains the iscS gene and adjacent 500-bp flanking regions from the E. coli genome. These mutant plasmids were used to observe low copy effects when expressed in trans as well as to reintegrate the mutant iscS genes back into the chromosome of strain CL100 using previously described techniques (8). Chromosomal mutations were confirmed by amplification of iscS using IscS.No and IscS.Co primers (9) followed by sequence analysis.

Analysis of Growth Phenotype—Rich medium was LB and minimal medium was M9-supplemented with 0.2% glucose, 1 mM MgSO4, and leucine (40 µg/ml) unless otherwise stated (MC1061 is a leucine auxotroph). The following nutrients were added at the given concentrations when indicated: nicotinic acid (12.5 µg/ml), thiamin (1 µg/ml), isoleucine (40 µg/ml), and valine (40 µg/ml). Ampicillin was used at a final concentration of 100 µg/ml, and chloramphenicol was used at 20 µg/ml. To determine doubling times, cells from overnight cultures in LB were pelleted, washed once with minimal medium, resuspended, and inoculated (1:500) into LB. Cultures were incubated with shaking at 37 °C, and growth was measured by monitoring the absorbance at 600 nm. For assessment of nutritional requirements, cells were grown in nutrient broth, washed, and inoculated into minimal medium containing the relevant supplements prior to monitoring growth.

Isolation and Analysis of Unfractionated tRNA—Bulk tRNA was isolated as described previously (9) from 100 ml of uninduced cultures in LB supplemented with ampicillin (100 µg/ml). All of the cultures for tRNA analysis were harvested at mid-log phase (A600 = 0.5). Bulk gel-purified tRNA was quantitated by UV absorption at 260 nm, digested (100 µg) to nucleosides using nuclease P1 and alkaline phosphatase, and analyzed by high pressure liquid chromatography by the method of Gherke et al. (40) as described by us (9, 25). All of the thionucleosides were analyzed at 260 nm with the exception of 4-thiouridine, which was also analyzed at 330 nm (data not shown). To account for variation in sample loading, the thiouridines and s2C were quantitated by taking the ratio of each peak area to that of pseudouridine in the same chromatogram (41). The levels of ms2i6A were taken as a relative percentage of the sum of ms2i6A and i6A, which should be independent of the amount of tRNA loaded onto the column. The values were then reported relative to the wild type, which is set to 1.0 (Table I).


View this table:
[in this window]
[in a new window]
  		TABLE I
Characterization of chromosomal iscS mutants

Levels of the indicated thionucleosides are presented as the ratio of peak area to that of pseudouridine with the level in MC1061 set to 1.0. Errors were based on duplicate runs and are <=15%. Doubling time (t2) is for growth in LB at 37 °C. SDH and 6-phosphogluconate dehydratase (PGD) activities each represent the mean ± S.D. of duplicate experiments. SDH activity is reported relative to WT. PGD activity is expressed as units/milligram protein in extracts where 1 U = 1 µmol/min. ND, not determined.

 
Western Analysis of iscS Expression—Antiserum to IscS was prepared from a single rabbit by Bethyl Laboratories (Montgomery, TX). Affinity-purified serum was obtained by passing the serum in 0.5 M NaCl through a column of consisting of 50 mg of IscS immobilized on CNBr-activated Sepharose 4B beads (Amersham Biosciences). Antiserum was eluted from the column using 50 mM glycine (pH 3.0) followed by immediate neutralization with 0.5 M Tris-HCl (pH 8.0). For Western analysis, cultures were grown to mid-log phase (A600 0.5) in LB medium, lysed by French Press, and separated into soluble and insoluble fractions by centrifugation at 30,000 x g for 15 min. Samples were subjected to SDS-PAGE analysis (42) using 12% gels, and immunoblotting was carried out using enhanced chemiluminescence detection (Amersham Biosciences). A 1:500 dilution of rabbit anti-IscS affinity-purified antiserum was followed by a 1:10,000 dilution of an anti-rabbit horseradish peroxidase conjugate.

35S Transfer Assays—Persulfide transfer activity was determined as described previously (21) with the following modifications. Assays were carried out at 23 °C for 30 s and contained 1 µM IscS, 18.5 µM IscU, 50 mM Hepes (pH 7.3), 150 mM KCl, and 10 mM MgCl2 in a final volume of 35 µl. Reactions were initiated by the addition of L-[35S]cysteine (0.19 Ci/mmol; Amersham Biosciences) to a final concentration of 57 µM and were terminated by centrifugation at 1000 x g through a size-exclusion column, Bio-Gel P-6 column (Bio-Rad). The spin column eluant was mixed with SDS-PAGE sample loading buffer to give final concentrations of 1% glycerol, 5 mM Tris (pH 8.0), and 0.001% bromphenol blue. Samples were immediately analyzed by gel electrophoresis using a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate in the absence of added reducing agent. 35S was visualized by exposure on a phosphorimaging screen and analyzed using a Personal Molecular Imager FX (Bio-Rad) and analyzed with Quantity One quantitation software supplied with the instrument.

Assembly of Fe-S Clusters on IscU—IscS-mediated assembly of iron-sulfur clusters on IscU was assayed as described previously (21). Reaction mixtures contained 2 µM IscS, 50 µM IscU, 2 mM ferric citrate, and 10 µM pyridoxal phosphate in 50 mM Hepes (pH 7.3), 150 mM KCl, and 10 mM MgCl2. Cluster assembly was initiated by the addition of 2.5 mM L-cysteine and 5 mM dithiothreitol and was monitored by circular dichroism on a Jasco J-720 spectropolarimeter.

Enzyme Assays
Cysteine Desulfurase—Cysteine desulfurase activity of IscS proteins was performed by monitoring sulfide release from L-cysteine as described previously (19, 21) and quantifying the sulfide in the reaction mixture by the methylene blue procedure of Segel (43). Specific activity of the purified proteins was determined at a cysteine concentration of 2.5 mM. Protein concentration was 100 nM.

Succinate Dehydrogenase—In replicates of six, cultures (5 ml) were grown in LB medium to mid-log phase (A650 = 0.6). Cell pellets from 3 ml of culture were frozen at –20 °C and assayed within 1 week. Pellets were resuspended and washed in 300 µl of 20 mM Tris citrate buffer (pH 8) and assayed as described previously (12).

6-Phosphogluconate Dehydratase—Cultures were grown in minimal A salts supplemented with 0.2% sodium gluconate and 0.2% casamino acids as well as with leucine, isoleucine, nicotinate, and thiamin at the levels described above. Cells were harvested at A600 = 0.8 by centrifugation at 8000 x g for 10 min. After storage at –80 °C, cells were resuspended in 50 mM Tris (pH 7.5), incubated with lysozyme for 20 min on ice, sonicated (3 x 20-s bursts), and then centrifuged in a microcentrifuge for 5 min. The supernatant was removed and immediately frozen on dry ice and stored until assayed (<24 h). Dehydratase was assayed in two steps for the formation of pyruvate according to the procedure described by Gardner and Fridovich (44). Activity is expressed in units/milligram protein where 1 unit = 1 µmol/min pyruvate formed.

s4U Assays—DEAE filter disc assays were performed as previously described for the formation of s2U (30) and s4U (27). For s4U, the assay mixture contained 50 mM Tris (pH 7.5), 50 mM KCl, 5 mM MgCl2, 4 mM ATP, 20 µM unmodified tRNAPhe, 20 µM L-[35S]cysteine, 300 nM ThiI, and 1 µM His6-tagged mutant or WT IscS. Reaction time was 8 min at 37 °C. Assays for s2U formation were performed similarly using unmodified tRNAGlu as substrate (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of IscS Mutants—Alanine-scanning mutagenesis was performed on a 14 amino acid region (Ser323-Ser336) that encompasses the active site loop region of E. coli IscS. Fourteen plasmids containing the relevant alanine substitutions in IscS were generated (see "Materials and Methods"). The plasmids were confirmed by sequencing and transformed into CL100(DE3) where the plasmid-encoded gene provided the only source of IscS in the cell. Alignment of this region from a selection of group I IscS homologs from taxonomically diverse organisms is shown in Fig. 3.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 3.
Sequence alignment of the putative active site loop region of selected IscS homologs. IscS homologs from a wide taxonomic range of organisms are represented. The top sequence is from E. coli IscS and is the basis for the numbering. The region of E. coli IscS mutated in this study (Ser323-Ser336) is shown in boldface, and mutated residues chosen for further study are capitalized as is the active site cysteine, Cys328. The regions of the active site loops of E. coli IscS (37) and T. maritima (36) that were disordered in their crystal structure are underlined. Abbreviations used are EC, E. coli; TM, T. maritima; BS, Bacillus subtilis; HS, Homo sapiens; SC, S. cerevisiae; CE, Caenorhabditis elegans; AF, Archaeoglobus fulgidus; AA, Aquifex aeolicus; YP, Yersinia pestis; MT, Methanococcus thermoautotropicus; AN, Anabena sp.; PA, Pseudomonas aerogenosa; TV, Trichomonas vaginalis; ML, Mycobacterium leprae.

 
Thionucleoside Profile of iscS Mutants—As an initial screen for mutant IscS function, thionucleoside levels in each of the 14 plasmid-containing strains were determined by high pressure liquid chromatography analysis of tRNA enzymatic digests. The strain containing the C328A mutant had a thionucleoside profile identical to the iscS{Delta} strain, consistent with previous reports that Cys328 is a critical residue in IscS. Two of the 13 remaining mutations (S326A and L333A) resulted in altered thionucleoside levels. These alleles and two additional mutations as controls (S323A and S336A) were integrated into the chromosome for further analysis. The levels of each of the four tRNA thionucleosides isolated from the four resulting mutants are shown in Table I. The two control mutants (S323A and S336A) had thionucleoside levels indistinguishable from those of a wild-type (MC1061) strain. In contrast, the S326A and L333A mutants had significantly decreased levels of s2C and ms2i6A while maintaining wild-type levels of the thiouridines mnm5s2U and s4U. The spectrum of thionucleosides of the S326A chromosomal mutant is shown in Fig. 4. A similar spectrum was observed with the L333A mutant. The mutant spectra are different than either the parental or {Delta}iscS strains being instead similar to that produced by a block in miaB (Fig. 1) (32), consistent with the MiaB-catalyzed step being defective in the iscS mutants. MiaB is a member of the SAM radical superfamily of proteins that contain an oxygen labile Fe-S cluster (33). Thus, for this thionucleoside in particular, the data support the conclusion that an Fe-S cluster protein is defective.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4.
High pressure liquid chromatography thionucleoside profile of parent strain MC1061 (A), {Delta}iscS strain CL100 (B), and iscS S326A chromosomal mutant (C). Mutants lacking ms2i6A display an increased peak that represents the precursor i6A. Similarly, mutants lacking mnm5s2U contain mnm5U.

 
Growth Is Compromised in iscS Mutants—E. coli strains lacking iscS have well documented growth defects, including slow growth in rich medium. Growth properties of the four iscS mutants were explored. The doubling time of these mutant strains in rich medium are listed in Table I. The two mutants, L333A and S336A, that were defective in thionucleoside production had a longer doubling time than the other two point mutants or the wild-type strain. However, in both cases, the doubling time was significantly shorter than that of an {Delta}iscS mutant. In M9 minimal media supplemented with glucose and leucine (to satisfy parental auxotrophy), strain CL100 ({Delta}iscS) required nicotinic acid, thiamine, and isoleucine for growth (9). Under the same conditions, the S326A and L333A mutants grew in the absence of nicotinic acid, thiamine, or isoleucine after a significant lag. The lag time could be shortened by isoleucine or thiamine. The control S323A and S336A mutants exhibited growth behavior similar to the parent strain. In summary, the S326A and L333A mutants differ from the remaining alanine mutants and from the iscS null mutant in growth properties as they did in the thionucleoside analysis. To more directly test the status of in Fe-S clusters in the S326A and L333A mutants, succinate dehydrogenase and phosphogluconate dehydratase levels were measured in cell-free extracts. These data, presented in Table I, showed that SDH activity in the S326A and L333A point mutants was impaired to a similar extent as in the iscS deletion strain, whereas the dehydratase level is also consistent with defects in these two mutants.

iscS (L333A and S326A) Mutant Phenotypes Are Similar to Those of Null Mutants in iscAU—Of the genes in the isc operon, iscS is uniquely required for sulfur transfer to other proteins (e.g. ThiI) in addition to its role in Fe-S cluster synthesis. The data above were consistent with mutants L333A and S326A being defective solely in the function of IscS required for Fe-S biosynthesis, predicting that mutants defective in the downstream isc genes would have a similar phenotype. Results from the analysis of two additional isc mutant strains were consistent with this hypothesis. The first strain, DM5420, is a S. enterica mutant described previously (12) carrying a polar insertion in iscA. The second, OD1190, is an E. coli strain that carries a polar mutation in iscU. Data in Fig. 5 show that tRNA isolated from the iscU mutant contained no ms2i6Aors2C(<0.01) and thiouridines mnm5s2U and s4U were unaffected (1.0). Similar results were found for strain DM5420 (data not shown). In addition, as reported previously (12) and confirmed herein, the SDH activity in DM5420 was ~35% that of the wild-type strain. In summary, by every assay used (growth, SDH activity, thionucleoside accumulation), the L333A,S326A mutants were more similar to the iscA and iscU mutants than the {Delta}iscS mutant.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
High pressure liquid chromatography thionucleoside profile of E. coli WT strain MG16555 (A) and {Delta}iscU mutant strain OD1190 (B) after extended growth (24 h).

 
The iscS Mutations Do Not Reduce Protein Levels—To eliminate the possibility that the phenotype of the two iscS mutants was a result of decreased amounts of the mutant proteins, levels of IscS in the parent and chromosomal mutant strains were measured by immunoblotting. Fig. 6 shows a comparison of the levels of IscS in cells grown to mid-log phase (A600 = 0.6) in rich medium (LB). Mutant protein was found in the insoluble fraction of cell lysates, but each of the mutant strains accumulated levels of soluble IscS greater than the parent strain. This finding, together with the cysteine desulfurase activity studies described below, suggest that the phenotypes observed did not result from reduced levels of the mutant proteins.



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 6.
Western analysis of expression levels of IscS in wild type and mutant strains harvested at mid-log phase. Panel A shows L333A and S326A mutants and parent strain MC1061. Panel B shows {Delta}iscS strain CL100, S336A mutant, and IscS standard. Each lane contained 5 µg of protein. Sol and Pel refer to soluble fraction and cell pellet, respectively.

 
In Vitro Activity of Mutant IscS Proteins—The above results suggest that the S326A and L333A IscS mutants have a defect in Fe-S synthesis in vivo. To correlate this defect with a specific biochemical function of IscS, in vitro assays were used to measure the activity of IscS. IscS from the four mutants described in Table I was purified as the N-terminal His6 fusion protein. Table II shows the results of three enzymatic activity assays with these forms as well as that of a C328A mutant. As anticipated, the C328A mutant is essentially devoid of cysteine desulfurase activity and activity in s4U and s2U synthesis. Each of the other active site loop mutant proteins exhibited cysteine desulfurase activity that was equal to or greater than that of the wild-type enzyme. Furthermore, an in vitro assay of s4U biosynthesis using 35S-labeled cysteine showed that each mutant protein (>=1 µM) was able to transfer the sulfur of cysteine to the appropriate tRNA substrate with similar efficiency, although the S326A mutant was consistently ~30–40% lower at all of the concentrations tested (0.2–1 µM). All of the mutant proteins showed s2U synthesis activity that was not significantly different from wild type at all of the concentrations tested. These data showed that the inability of the S326A and L333A mutants to build Fe-S clusters in vivo was not the result of lowered cysteine desulfurase activity.


View this table:
[in this window]
[in a new window]
  		TABLE II
In vitro assays of wild-type and mutant IscS proteins

All of the assays used purified proteins with the exception of the C328A form that was expressed in strain CL100(DE3) and was ~40% pure. Assay procedures are as described under "Materials and Methods."

 
IscS Mutants Are Proficient to Transfer Sulfur to IscU—The S326A and L333A mutant IscS proteins were assayed for their ability to transfer sulfur to IscU. The assay employed monitors IscS-catalyzed transfer of [35S]sulfur from L-[35S]cysteine to IscU using nonreducing SDS-PAGE (21). Fig. 7 shows that each of the IscS mutants was labeled to a similar extent to wild-type IscS and that each was able to transfer the labeled sulfur to IscU with efficiency similar to wild type IscS. These results indicate that replacement of Ser326 and Leu333 with alanine does not block sulfur transfer to IscU. The similar extent of labeling of the band corresponding to an IscS-IscU heterodimer further suggests that these substitutions do not have dramatic effects on complex formation between IscS and IscU.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 7.
Sulfur transfer activities of IscS mutants. L-[35S]Cysteine was incubated for 30 s with wild-type or mutant forms of His-tagged IscS (1 µM) in the absence or presence of IscU (18.5 µM), separated on spin column, and subjected to non-reducing SDS-PAGE. 35S label incorporation was analyzed with a PhosphorImager. Protein migration positions determined by Coomassie Blue staining are indicated on the left.

 
The ability of the S326A mutant to participate in assembly of iron-sulfur clusters on IscU was also investigated. Samples containing IscS, IscU, cysteine, and ferric chloride were incubated anaerobically in the presence of dithiothreitol, and cluster assembly was monitored by circular dichroism (21). Fig. 8 shows visible region CD spectra recorded at different times following cysteine addition for a reaction containing the S326A mutant. Maximal cluster formation was observed in ~30 min with some decay apparent at longer times. The CD spectrum is similar to that previously reported for wild-type IscS (21) with minima near 380 and 560 nm and a maximum near 430 nm. The inset shows that the initial rate of cluster formation for the S326A mutant is similar to or slightly greater than that of the wild-type parent. This result indicates that sulfur transfer to IscU mediated by the S326A mutant is effective in iron-sulfur cluster formation.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 8.
Fe-S cluster formation mediated by IscS(C326A). Reaction mixtures contained 2 µM IscS, 50 µM IscU, 2 mM ferric citrate, and 10 µM pyridoxal phosphate in 50 mM Hepes (pH 7.3), 150 mM KCl, and 10 mM MgCl2. Cluster assembly was initiated by the addition of 2.5 mM L-cysteine and 5 mM dithiothreitol. CD spectra were recorded from 500 to 680 nm at different times, and spectra after 0, 4, 6, 10, 16, and 26 min are shown. The spectrum from 350 to 700 nm recorded after 1 h reveals some decay of the cluster. The inset shows a comparison of the rate of cluster formation for the S326A mutant in this experiment with that for wild-type IscS monitored continuously at 560 nm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on the complex phenotype of E. coli iscS mutants and extensive in vitro work with purified protein, it is clear that IscS interacts with multiple protein sulfur acceptors. Here alanine scanning of the active site loop of E. coli IscS has been used to identify residues required for in vivo function of this protein. The data show that two substitutions, the highly conserved Ser326 and the nonconserved Leu333, selectively impair the role of IscS in the synthesis of Fe-S clusters. Analysis of thionucleoside accumulation served as an initial screen for IscS function. IscS is required for the biosynthesis of normal levels of all of the thionucleosides in E. coli (25) and S. typhimurium (26), although only two of them are expected to require Fe-S cluster enzymes in their synthesis. The finding that of the four thionucleosides, substitution mutants (S326A and L333A) were defective in synthesis of only s2C and ms2i6A indicates that the two roles of IscS have been separated. Decreased levels of the Fe-S cluster enzyme succinate dehydrogenase also support the conclusion that the S326A and L333A mutations result in a defect in Fe-S cluster biosynthesis in vivo.

Although these mutants were defective in Fe-S cluster biosynthesis in vivo, no in vitro defect was identified. The S326A and L333A mutant proteins were able to transfer sulfur to IscU in vitro. This experiment also shows that persulfide formation and stability are not detectably impaired in these mutants. Preliminary experiments that measure the rate of persulfide transfer to IscU for the S326A and L333A mutants show no significant differences with WT IscS.2 These results suggest that the role of IscS in Fe-S biosynthesis in vivo may be more complex than previously appreciated in that persulfide transfer to IscU is a necessary but not sufficient step.

Structural data from a crystal form of E. coli IscS shows that the loop containing the C328 can be positioned near the protein surface, greater than 17 Å from the buried pyridoxal phosphate cofactor (38). In both the T. maritima (37) and E. coli (38) structures, portions of this loop are disordered, consistent with its proposed flexibility. Neither Leu333 of E. coli IscS nor the equivalent of Glu329 in T. maritima NifS was observable in the crystal structure. Since Leu333 is not conserved, it may be required for interactions that are organism-specific. Unlike Leu333, Ser326 is resolved in the E. coli structure. The Ser326 serine hydroxyl is within hydrogen bonding distance to Arg39 of the other IscS monomer in the homodimer. However, Arg39 is not conserved (data not shown); therefore, this interaction may be of limited importance. Because Ser326 is highly conserved, the interactions in which it is required must also to some extent be conserved. Preliminary results from saturation mutagenesis of position 326 suggest that serine is the only acceptable amino acid at this position for normal growth.3 Aside from the Arg39 interaction, there is no obvious indication of the role of the serine based on the crystal structure. It may be required for a subtle modulation of persulfide reactivity or for protein-protein interaction with Isc proteins other than IscU. Future work will focus on the isolation of specific mutants defective in non Fe-S functions of IscS in an attempt to understand the structural basis of its sulfur transfer specificity.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM47296 (to D. M. D.), GM54264 (to L. E. V.), and GM57002 (to C. T. L.). Funds were also provided from a 21st Century Scientists Scholars Award from the J. M. McDonnell fund (to D. M. D.). 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

|| Supported by a Louis and Elsa Thomsen Wisconsin Distinguished Fellowship Award from the College of Agricultural and Life Sciences. Back

§ To whom correspondence should be addressed. Tel.: 608-262-3083; Fax: 608-262-3397; E-mail: clauhon{at}facstaff.wisc.edu.

1 The abbreviations used are: mnm5s2U, 5-methylaminomethyl-2-thiouridine; s4U, 4-thiouridine; s2C, 2-thiocytidine; ms2i6A, 6-dimethallyl-2-methylthioadenosine; SDH, succinate dehydrogenase; WT, wild type. Back

2 H. Urbina, and L. Vickery, unpublished observation. Back

3 E. Steenblock, J. S. Williams, and C. T. Lauhon, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mihara, H., and Esaki, N. (2002) Appl. Microbiol. Biotechnol. 60, 12–23[CrossRef][Medline] [Order article via Infotrieve]
  2. Marquet, A. (2001) Curr. Opin. Chem. Biol. 5, 541–549[CrossRef][Medline] [Order article via Infotrieve]
  3. Beinert, H. (2000) Eur. J. Biochem. 267, 5657–5664[Medline] [Order article via Infotrieve]
  4. Begley, T. P., Xi, J., Kinsland, C., Taylor, S., and McLafferty, F. (1999) Curr. Opin. Chem. Biol. 3, 623–629[CrossRef][Medline] [Order article via Infotrieve]
  5. Zheng, L., White, R. H., Cash, V. L., Jack, R. F., and Dean, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2754–2758[Abstract/Free Full Text]
  6. Zheng, L., Cash, V. L., Flint, D. H., and Dean, D. R. (1998) J. Biol. Chem. 273, 13264–13272[Abstract/Free Full Text]
  7. Kolman, C., and Soll, D. (1993) J. Bacteriol. 175, 1433–1442[Abstract/Free Full Text]
  8. Olson, J. W., Agar, J. N., Johnson, M. K., and Maier, R. J. (2000) Biochemistry 39, 16213–16219[CrossRef][Medline] [Order article via Infotrieve]
  9. Lauhon, C. T., and Kambampati, R. (2000) J. Biol. Chem. 275, 20096–21003[Abstract/Free Full Text]
  10. Schwartz, C. J., Djaman, O., Imlay, J. A., and Kiley, P. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9009–9014[Abstract/Free Full Text]
  11. Tokumoto, U., and Takahashi, Y. (2001) J. Biochem. 130, 63–71[Abstract/Free Full Text]
  12. Skovran, E., and Downs, D. M. (2000) J. Bacteriol. 182, 3896–3903[Abstract/Free Full Text]
  13. Mihara, H., Kurihara, T., Yoshimura, T., Soda, K., and Esaki, N. (1997) J. Biol. Chem. 272, 22417–22424[Abstract/Free Full Text]
  14. Mihara, H., Kurihara, T., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 127, 559–567[Abstract/Free Full Text]
  15. Mihara, H., Kato, S., Lacourciere, G. M., Stadtman, T. C., Kennedy, R. A., Kurihara, T., Tokumoto, U., Takahashi, Y., and Esaki, N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6679–6683[Abstract/Free Full Text]
  16. Zheng, M., Wang, X., Templeton, L. J., Smulski, D. R., LaRossa, R. A., and Storz, G. (2001) J. Bacteriol. 183, 4562–4570[Abstract/Free Full Text]
  17. Takahashi, Y., and Tokumoto, U. (2002) J. Biol. Chem. 277, 28380–28383[Abstract/Free Full Text]
  18. Zheng, L, White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714–4720[CrossRef][Medline] [Order article via Infotrieve]
  19. Flint, D. H. (1996) J. Biol. Chem. 271, 16068–16074[Abstract/Free Full Text]
  20. Smith, A. D., Agar, J. N., Johnson, K. A., Frazzon, J., Amster, I. J., Dean, D. R., and Johnson, M. K. (2001) J. Am. Chem. Soc. 123, 11103–11104[CrossRef][Medline] [Order article via Infotrieve]
  21. Urbina, H. D., Silberg, J. J., Hoff, K. G., and Vickery, L. E. (2001) J. Biol. Chem. 276, 44521–44526[Abstract/Free Full Text]
  22. Kato, S., Mihara, H., Kurihara, T., Takahashi, Y., Tokumoto, U., Yoshimura, T., and Esaki, N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5948–5952[Abstract/Free Full Text]
  23. Tokumoto, U., Nomura, S., Minami, Y., Mihara, H., Kato, S., Kurihara, T., Esaki, N., Kanazawa, H., Matsubara, H., and Takahashi, Y. (2002) J. Biochem. 131, 713–719[Abstract/Free Full Text]
  24. Frazzen, J., and Dean, D. R. (2003) Curr. Opin. Chem. Biol. 7, 166–173[CrossRef][Medline] [Order article via Infotrieve]
  25. Lauhon, C. T. (2002) J. Bacteriol. 184, 6820–6829[Abstract/Free Full Text]
  26. Nilsson, K., Lundgren, H. K., Hagervall, T. G., and Bjork, G. R. (2002) J. Bacteriol. 184, 6830–6835[Abstract/Free Full Text]
  27. Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561–16568[CrossRef][Medline] [Order article via Infotrieve]
  28. Kambampati, R., and Lauhon, C. T. (2000) J. Biol. Chem. 275, 10727–10730[Abstract/Free Full Text]
  29. Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283–8286[Abstract/Free Full Text]
  30. Mueller, E. G., Palenchar, P. M., and Buck, C. J. (2001) J. Biol. Chem. 276, 33588–33595[Abstract/Free Full Text]
  31. Kambampati, R., and Lauhon, C. T. (2003) Biochemistry 42, 1109–1117[CrossRef][Medline] [Order article via Infotrieve]
  32. Esberg, B., Leung, H. C. E., Tsui, H. C. T., Bjork, G. R., and Winkler, M. E. (1999) J. Bacteriol. 181, 7256–7265[Abstract/Free Full Text]
  33. Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Nucleic Acids Res. 29, 1097–1106[Abstract/Free Full Text]
  34. Pierrel, F., Bjork, G. R., Fontecave, M., and Atta, M. (2002) J. Biol. Chem. 277, 13367–13370[Abstract/Free Full Text]
  35. Leipuviene, R., Qian, Q., and Bjork, G. R. (2004) J. Bacteriol. 186, 758–766[Abstract/Free Full Text]
  36. Jager, G., Leipuviene, R., Pollard, M. G., Qian, Q., and Bjork, G. R. (2004) J. Bacteriol. 186, 750–757[Abstract/Free Full Text]
  37. Kaiser, J. T., Clausen, T., Bourenkow, G. P., Bartunik, H. D., Steinbacher, S., and Huber, R. (2000) J. Mol. Biol. 297, 451–464[CrossRef][Medline] [Order article via Infotrieve]
  38. Cupp-Vickery, J. R., Urbina, H., and Vickery, L. E. (2003) J. Mol. Biol. 330, 1049–1059[CrossRef][Medline] [Order article via Infotrieve]
  39. Link, A. J., Phillips, D., and Church, G. M. (1997) J. Bacteriol. 179, 6228–6237[Abstract/Free Full Text]
  40. Gehrke, C. W., Kuo, K. C., McCune, R. A., and Gerhardt, K. O. (1982) J. Chromatogr. 230, 297–308[CrossRef][Medline] [Order article via Infotrieve]
  41. Buck, M., Connick, M., and Ames, B. N. (1983) Anal. Biochem. 129, 1–13[CrossRef][Medline] [Order article via Infotrieve]
  42. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  43. Siegel, L. M. (1965) Anal. Biochem. 11, 126–132[CrossRef][Medline] [Order article via Infotrieve]
  44. Gardner, P. R., and Fridovich, I. (1991) J. Biol. Chem. 266, 1468–1473
  45. Xi, J., Ge, Y., Kinsland, C., McLafferty, F. W., and Begley, T. P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 8513–8518[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Microbiol. Mol. Biol. Rev.Home page
C. Ayala-Castro, A. Saini, and F. W. Outten
Fe-S Cluster Assembly Pathways in Bacteria
Microbiol. Mol. Biol. Rev., March 1, 2008; 72(1): 110 - 125.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
P. C. Dos Santos, D. C. Johnson, B. E. Ragle, M.-C. Unciuleac, and D. R. Dean
Controlled Expression of nif and isc Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems
J. Bacteriol., April 1, 2007; 189(7): 2854 - 2862.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
D. C. Johnson, M.-C. Unciuleac, and D. R. Dean
Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii
J. Bacteriol., November 1, 2006; 188(21): 7551 - 7561.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
O. Smid, E. Horakova, V. Vilimova, I. Hrdy, R. Cammack, A. Horvath, J. Lukes, and J. Tachezy
Knock-downs of Iron-Sulfur Cluster Assembly Proteins IscS and IscU Down-regulate the Active Mitochondrion of Procyclic Trypanosoma brucei
J. Biol. Chem., September 29, 2006; 281(39): 28679 - 28686.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
H. K. Lundgren and G. R. Bjork
Structural Alterations of the Cysteine Desulfurase IscS of Salmonella enterica Serovar Typhimurium Reveal Substrate Specificity of IscS in tRNA Thiolation.
J. Bacteriol., April 1, 2006; 188(8): 3052 - 3062.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Shigi, T. Suzuki, T. Terada, M. Shirouzu, S. Yokoyama, and K. Watanabe
Temperature-dependent Biosynthesis of 2-Thioribothymidine of Thermus thermophilus tRNA
J. Biol. Chem., January 27, 2006; 281(4): 2104 - 2113.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
E. Skovran, C. T. Lauhon, and D. M. Downs
Lack of YggX Results in Chronic Oxidative Stress and Uncovers Subtle Defects in Fe-S Cluster Metabolism in Salmonella enterica
J. Bacteriol., November 15, 2004; 186(22): 7626 - 7634.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/19/19551    most recent
M401261200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend