HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 39, 40505-40510, September 24, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/39/40505    most recent M403985200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martinez-Gomez, N. C. Articles by Downs, D. M. Search for Related Content PubMed PubMed Citation Articles by Martinez-Gomez, N. C. Articles by Downs, D. M. Social Bookmarking                      What's this?

Mutational Analysis of ThiH, a Member of the Radical S-Adenosylmethionine (AdoMet) Protein Superfamily^*

Norma C. Martinez-Gomez, Matt Robers, and Diana M. Downs

From the Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53726-4087

Received for publication, April 9, 2004 , and in revised form, July 1, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thiamine pyrophosphate (TPP) is an essential cofactor for all forms of life. In Salmonella enterica, the thiH gene product is required for the synthesis of the 4-methyl-5- hydroxyethyl-thiazole monophosphate moiety of TPP. ThiH is a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins that is characterized by the presence of oxygen labile [Fe-S] clusters. Lack of an in vitro activity assay for ThiH has hampered the analysis of this interesting enzyme. We circumvented this problem by using an in vivo activity assay for ThiH. Random and directed mutagenesis of the thiH gene was performed. Analysis of auxotrophic thiH mutants defined two classes, those that required thiazole to make TPP (null mutants) and those with thiamine auxotrophy that was corrected by either L-tyrosine or thiazole (ThiH* mutants). Increased levels of AdoMet also corrected the thiamine requirement of members of the latter class. Residues required for in vivo function were identified and are discussed in the context of structures available for AdoMet enzymes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thiamine pyrophosphate is an essential cofactor that stabilizes acyl carbanions generated by several enzymes in carbohydrate metabolism such as transketolase, -ketoacid decarboxylase, -ketoacid dehydrogenase, and acetolactate synthase. The biosynthesis of thiamine pyrophosphate involves the separate formation of the 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP)¹ and thiazole monophosphate (THZ-P) moieties (Fig. 1). These moieties are then coupled and phosphorylated, forming thiamine pyrophosphate (1). In Escherichia coli and Salmonella at least seven genes, thiFSGH, thiI, iscS, and dxs, are required to convert 1-deoxy-D-xylulose phosphate, L-cysteine and L-tyrosine to THZ-P (2–4). Many of the mechanistic aspects of this conversion have been worked out in vitro (5–8). Our understanding of thiazole biosynthesis has increased dramatically with the reconstitution of the pathway in cell-free extracts by two groups (9, 10). Although these groups used proteins from Bacillus subtilis and E. coli, the proposed mechanisms are similar. The primary difference between these pathways is the generation of the glycine imine intermediate from which the C-2 and N-3 of the thiazole product are derived. In B. subtilis, ThiO catalyzes the oxidation of glycine to form the imine (11), whereas in E. coli, the imine intermediate is proposed to be the product of ThiH (10). In the case of ThiH, it is suggested that an interaction between S-adenosylmethionine (AdoMet), and the [Fe-S] cluster allows the formation of the 5'-deoxyadenosyl radical, generating a tryosyl radical that results in the glycine imine via a series of steps (10).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Biosynthesis of thiamine pyrophosphate in Salmonella enterica. Gene products involved in each reaction are indicated next to the relevant arrows. The specific reaction catalyzed by ThiG and ThiH in Salmonella have not been defined. A mechanism to generate THZ-P has been proposed (10) as shown: the deoxyadenosyl radical (DOA·) generates a tyrosinyl radical 2 that with conformational changes prior to cleavage generates the corresponding quinone methide 3 and a glycinyl radical 4. It is not well understood which electron acceptor will allow this radical to lose an electron, but an imminium ion will be formed after this reaction 5. The condensation between 1-deoxy-D-xylulose phosphate, the thiocarboxylate, and the imminium ion will occur leading to the formation of an intermediate that is believed to be the substrate of ThiG 6. Condensation will be achieved, and THZ-P would be generated. The formation of HMP is shown as well; the reaction involves a complex rearrangement of 5-aminoimidazole ribotide to generate 7. ThiC is the only required gene product that has been identified to catalyze this reaction. ThiD catalyzes the phosphorylation of 7, generating HMP-PP. Condensation between THZ-P and HMP-PP occurs with the activity of ThiE prior to phosphorylation by ThiL generating thiamine pyrophosphate.

ThiH homologs are not prevalent in genomes of microorganisms that synthesize thiamine. Organisms that are able to synthesize thiamine yet lack thiH (e.g. B. subtilis and Rizobium etli) have thiO, which encodes a glycine oxidase (11, 21). The remaining genes involved in thiamine synthesis are more widely conserved (22, 23). The difference(s) between the chemistries and metabolic requirements of the ThiO and ThiH pathways in vivo are not clear.

Our interest in thiazole biosynthesis focuses on understanding the integration of this pathway with the metabolic network of the cell. The work herein was initiated to better understand both the direct and indirect requirements for ThiH activity in vivo. A series of thiH mutations were generated, and the mutant phenotypes were characterized. Data obtained support a role for an [Fe-S] cluster and AdoMet in the function of ThiH and define other critical residues that suggest structural features of the protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains, Media, and Chemicals
The strains used in this study are derived from S. enterica LT2 and are listed with their respective genotypes in Table I. Minimal media was NCE (24) supplemented with MgSO₄ (1 mM) and glucose (11 mM). The final concentrations of L-tyrosine, and thiamine were 0.1 mM and 400 nM, respectively. Rich media was Difco nutrient broth (8 g/liter) with NaCl (5 g/liter). Solid media contained 1.5% agar. The final concentrations of antibiotics were: chloramphenicol, 20 µg/ml; ampicillin, 40 µg/ml; kanamycin, 50 µg/ml. All chemicals were purchased from Sigma Chemical Co. 4-Amino-5-hydroxymethyl-2-methylpyrimidine was purified from spent medium as described previously (25) and was provided where indicated.

PCR Mutagenesis—For PCR mutagenesis plasmid pThiH1 (wild type thiH in pSU19 (26)) was used as a template. Gene specific primers (Integrated DNA Technologies, Coralville, IA) with the following sequence were used in amplification reactions with Vent (exo-) polymerase (New England Biolabs, Beverly, MA): NdeF3P, 5'PO₄GAAGGAGATATACATATGAAAACCTTCACCGAC-3' and BamR2P, 5'PO₄AGGATCCTCACCGCGTTTGCGAAG-3'. The mutagenized population of thiH fragments was gel purified and ligated into pSU19 that had been digested with SmaI and treated with calf intestinal phosphatase (Promega, Madison, WI). Ligation mixtures were transformed into strain DM2275 (thiH) and plated on nutrient broth-chloramphenicol. The chloramphenicol resistant transformants were scored for their nutritional requirement by replica printing to minimal media, minimal media with L-tyrosine, and minimal media with thiamine. All plasmid-containing strains were reconstructed, and the phenotypes were verified.

Site-directed Mutagenesis—Two different methods for site-directed mutagenesis were used. One method involved the generation of two PCR products using either Nde3P or BamR2P primers with a primer with the desired mutation. Both fragments were gel purified and used together in a PCR reaction with the external Nde3P and BamR2P primers, generating a full-length thiH that was then ligated into pSU19. A second method was performed as described in the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutant plasmids were confirmed by sequence analysis.

DNA Sequencing—DNA sequencing was performed at the University of Wisconsin-Madison Biotechnology Center-Nucleic Acid Facility. DNA sequence analysis program BLAST was used to compare the sequences to wild type thiH sequence from the data base (27).

Phenotypic Analysis
Liquid Growth—Nutritional requirements were determined by replica printing on solid media and liquid growth analysis. For the latter, strains were grown overnight in nutrient broth (plus chloramphenicol as indicated) and cells were pelleted, resuspended in saline, and inoculated into liquid media, and absorbance at 650 nm (A₆₅₀) was used to monitor growth (25). When final yield was to be measured, growth was initiated as described above, and the A₆₅₀ was measured after 18 h of incubation at 37 °C with shaking. When strains containing gshA, apbC, or apbE were assessed for thiazole synthesis, HMP was provided in the media at a level that allowed growth of a thiC mutant.

Overexpression of AdoMet Synthase—Plasmids pMETK2 and a control vector (pPL1 (28)) were a gift from N. Buan and J. C. Escalante-Semerena. MetK was induced by the addition of isopropyl--D-thiogalactoside to the medium at a final concentration of 1 mM.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Conserved [Fe-S] Cluster and AdoMet Binding Motifs Are Critical for ThiH Function in Vivo—The thiH gene was mutagenized using both random (PCR) and directed approaches. Missense mutations in thiH were generated in plasmid pThiH1 and introduced into a strain lacking a functional thiH gene in the chromosome. Substitutions that affected the in vivo function of ThiH were identified by growth phenotype. The locations of mutated residues are shown in Fig. 2A, and the specific substitutions are listed in Fig. 2B.

View larger version (45K): [in this window] [in a new window]   FIG. 2. Position and identity of thiH mutant alleles. A, the residue affected by thiH alleles is shown in the context of the primary structure. Substitutions of residues shown with a black background result in a null phenotype, and substitutions shown in light gray result in a ThiH* phenotype as described in the text. Changes in the circled residues generated either a ThiH* or thiH null phenotype depending on the substitution. Residues that define the binding sites predicted by Sofia et al. (13) for the [Fe-S] cluster and AdoMet are boxed. B, specific substitutions found at each mutant residue are shown. Substitutions in the left box result in a thiH null phenotype, and those in the right box generate a ThiH* phenotype. Fractions in parentheses represent the proportion of included homologs that maintain the same residue as S. enterica for each position. Homologs were considered from E. coli, Campilobacter, Clostridium, Shewanella, Vibrio, Yersinia, and Desulfovibrio species. Substitutions marked with an asterisk were the result of site-directed mutagenesis. Mutants containing two substitutions are not reflected in the figure.

Effect of Mutations Affecting the Putative AdoMet Binding Motif—S. enterica ThiH contains a sequence block (LLLVTGEHQAKV¹³⁶) that shares weak identity with the AdoMet binding motif found in S-adenosylmethionine methyltransferases (13). In this motif, residues Gly-130 and Glu-131 are conserved among the 20 ThiH homologs considered in this analysis. Alanine substitutions at G130A or E131A inactivated ThiH. In addition, an E131K substitution mutation was isolated in a screen for randomly generated for thiH null alleles.

Critical Residues Are Dispersed Throughout ThiH—Random mutagenesis identified amino acid substitutions that eliminated in vivo function of ThiH. Only alleles that resulted in stable proteins (as determined by immunoblot analysis) were considered. Residues critical for function were distributed throughout the protein (Fig. 2A). As expected, substitutions that eliminated in vivo function of the enzyme primarily changed conserved or invariant residues; a notable exception was the E187D substitution. At this position, a glutamic acid residue is found in only 3 of 20 ThiH homologs, and yet in S. enterica ThiH reduction of the side chain of this residue by one methylene group generated a null phenotype. The null phenotype of this conservative substitution was confirmed by creating the same allele by site-directed mutagenesis.

Multiple Residue Changes That Generate a ThiH* Phenotype—We identified previously a unique class of thiH alleles that encode proteins (referred to as ThiH*) that are functional in vivo when tyrosine is present (12). The mutations of this class described previously generated mutant proteins ThiH^H124Y or ThiH^P347S (12). Additional plasmids encoding thiH alleles of this class were generated in this work (Fig. 2).

Substitution of Residues That Result in a Null or ThiH* Phenotypes—Two residues, Cys-285 and Ala-61 (Fig. 2), were of interest because substitutions at these residues were found in proteins that caused null or ThiH* phenotypes. In the case of residue Cys-285, a change to histidine eliminated function, whereas a change to an alanine resulted in the ThiH* phenotype. Strikingly, only 11 of 20 homologs have a cysteinyl residue at this position, and the remaining 9 have an alanine.

An alanine to threonine change at position 61 resulted in a null phenotype, whereas the ThiH* phenotype was observed with a change to serine and only when another mutation was present, P334Q. thiH alleles with either one of these single substitutions encoded functional proteins.

AdoMet Synthase in Multicopy Restores Thiamine Synthesis to thiH* Mutants—We noted that the H124Y mutation was juxtaposed to the AdoMet binding motif (Fig. 2A), raising the possibility that a ThiH* phenotype reflected weak AdoMet binding. To pursue this hypothesis, the wild type allele of the gene encoding S-adenosylmethionine synthase (metK) was introduced into two mutant strains, DM4104 and DM4106, that have a ThiH* phenotype and contain ThiH^H124Y and ThiH^P347S mutant proteins, respectively. The resulting strains were monitored for thiamine-independent growth. As shown in Fig. 3, increased levels of MetK restored thiamine synthesis in these two strains. The induction of metK in plasmid pMETK2 increases AdoMet synthase activity >5-fold.²

View larger version (17K): [in this window] [in a new window]   FIG. 3. Increased AdoMet synthase activity restores thiamine synthesis to thiH* mutants. Strains were grown at 37 °C in minimal glucose medium as described under "Experimental Procedures," and growth was monitored over time as A650. Strains shown are: A, DM4104 (ThiHH124Y); B, DM4106 (ThiHP347S); and C, DM2275 (thiH::Tn10). Each panel shows data from the relevant strain with plasmid pMETK2 (triangles) and with an empty vector (squares) in minimal medium (open symbols). Filled symbols represent growth in the presence of 100 nM thiamine.

ThiH* Phenotype Caused by Other Metabolic Functions Is Distinct from That of thiH Alleles—Mutations in loci involved in Fe-S cluster metabolism (gshA, apbC, apbE, iscA) result a ThiH* phenotype (12, 18, 19). The induction of metK in gshA, apbC, or apbE mutant strain backgrounds failed to allow significant growth in the absence of tyrosine or THZ (data not shown). This result suggested that the ThiH* phenotype could be generated by at least two mechanisms. This interpretation was consistent with the observation that suppressors of the gshA, apbC, or apbE mutant strains (anaerobic growth, over-production of YggX) were unable to restore thiamine synthesis in strains with a thiH* allele (data not shown).

Plasmids containing each of nine alleles of thiH generating a ThiH* phenotype were transformed into gshA, apbE, or apbC mutant backgrounds. Growth of the resulting strains in medium supplemented with thiamine or L-tyrosine was assessed. The resulting data showed that each of the 27 resulting strains had a null mutant phenotype (i.e. requirement for THZ or thiamine). In medium with or without tyrosine, the cell density (A₆₅₀) of cultures of 26 of these strains was between 0.02 and 0.13 after 18 h of incubation with no further increase observed after 24 h of incubation. A culture of the remaining strain reached an A₆₅₀ of 0.3. In the presence of thiamine, all cultures reached high cell densities (A₆₅₀ > 0.7) after 18 h of incubation. Control experiments confirmed that a thiH null mutant (DM460) carrying each of these plasmids individually displayed a ThiH* phenotype, as reflected by a final A₆₅₀ ranging from 0.04 to 0.15 and 0.61 to 1.3 in minimal medium or minimal medium supplemented with tyrosine or thiamine, respectively. Finally, when the only source of ThiH was encoded by a plasmid carrying the wild type allele of thiH, strains with lesions in the gshA, apbE, and apbC genes retained a ThiH* phenotype (data not shown).

Oligomers Formed by Different ThiH* Mutant Proteins Restore Function—Four plasmids carrying thiH alleles encoding ThiH* proteins were transformed into a strain carrying a chromosomal thiH allele that encodes a ThiH* protein (DM4104). Growth of the resulting strains was assessed, and the results are summarized in Table II. Some combinations of the chromosomal and plasmid-encoded ThiH* proteins allowed thiamine-independent growth. Of the four alleles tested, three allowed growth on minimal medium when present in a strain containing the ThiH^H124Y mutant protein. A similar pattern of growth was seen with the same plasmids present in a strain containing the ThiH^P347S mutant protein (data not shown). As expected, strains carrying the same allele on the chromosome and plasmid maintained a requirement for thiazole or tyrosine. This result is consistent with (but not conclusive of) the functional form of ThiH being a multimer and does not exclude its interaction with other proteins as suggested (20).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structural Features Critical for ThiH Function—Sequence analysis of ThiH identifies a CXXCXXXC⁹² motif and residues of a weakly defined AdoMet binding motif (LLVTGEHQAKV¹³⁶) that together result in the assignment of ThiH to the AdoMet radical superfamily (13). The identification of substitutions at these residues that eliminate ThiH function support a role for AdoMet and the [Fe-S] cluster in enzyme mechanism. Among radical AdoMet proteins there is a near consensus requirement for an aromatic residue in the position adjacent to the third cysteinyl residue of this motif (e.g. X⁹¹). In S. enterica ThiH this residue is a tyrosine, and a substitution by alanine or phenylalanine inactivates the protein. In the published structures of biotin synthase (BioB) (15), coproporphyrinogen III oxidase (HemN) (16), and pyruvate-formatelyase activase this residue (Tyr, Phe, and Tyr respectively) interacts with the adenine moiety of AdoMet. In the case of BioB this interaction could be explained by a hydrogen bond between the hydroxyl group of the tyrosyl residue and the 3'-hydroxyl group of the adenyl moiety of AdoMet.³ The fact that phenylalanine cannot substitute for tyrosine at position 91 in the S. enterica ThiH suggests a critical role for the hydroxyl group of the tyrosine. In HemN and pyruvate-formate-lyase activase the aromatic interaction between the purine moiety of AdoMet and the respective phenylalanine residue is responsible for stabilization. Two residues within the putative AdoMet binding motif, namely Gly-130 and Glu-131, were found to be critical for function. Structural data available for HemN suggests that these residues interact with the adenine moiety of AdoMet.

Residue Glu-187 is critical for ThiH function in S. enterica. This may be a specific feature of a few ThiH homologs because a glutamyl residue is found at this position in only 3 of the 20 homologs analyzed. The structural role of residue Glu-187 is so critical that shortening the side chain of this residue by a single carbon eliminates enzyme activity. We predict that the Glu-187 residue is also critical for the function of the other two ThiH homologs in which it is present.

Comparison of the primary sequence of ThiH with those of radical AdoMet proteins, HemN and BioB, for which crystal structures are available identified a number of residues that are conserved in the active site. Residues Cys-89 and Tyr-91 are conserved in all radical AdoMet proteins (13). Our mutational analysis supports the important roles of these residues in enzyme function.

The ThiH* Phenotype Provides Insight on ThiH Structure and Function in Vivo—Mutants that require tyrosine, THZ, or thiamine are said to have a ThiH* phenotype. Our working model suggests that a ThiH* phenotype may result from disrupting one of two structural features of the ThiH protein, either AdoMet binding or [Fe-S] cluster integrity. We suggest that correction of the ThiH* phenotype by increased levels of AdoMet supports the idea of AdoMet stabilizing the former class of mutant proteins. The lesions in the two mutants that were suppressed by increased AdoMet levels are consistent with this hypothesis, in particular the H124Y substitution, which is directly adjacent to the AdoMet binding motif.

Mutants defective in apbC, apbE, or gshA functions are compromised in the assembly and/or repair of Fe-S clusters (12, 18), and thus we have proposed that the ThiH* phenotype in strains lacking these functions reflects a compromised Fe-S cluster (12). We propose that some thiH lesions that generate the ThiH* phenotype do so by disrupting the structure of the Fe-S cluster. The effect of such mutations would not be corrected by increased levels of AdoMet as is the case for mutations affecting AdoMet binding.

Significantly, tyrosine restores ThiH function in both classes of proteins suggesting that the binding of tyrosine may provide stability lost by reduced binding of AdoMet or a destabilized Fe-S cluster. There is precedent for a stabilizing role of substrate in radical AdoMet enzymes. In the case of BioB there is >20-fold enhancement of AdoMet binding when the substrate (dethiobiotin) is bound (15). Noteworthy was the finding that tyrosine did not restore function to a ThiH protein that was simultaneously compromised in both AdoMet and Fe-S cluster binding, perhaps indicating the severity of the structural defects in this situation.

Analysis of the BioB and HemN structures show that the C-terminal portion of the protein is partially folded back with residues of the N-terminal in close proximity. We envision an active site of ThiH that is defined by residues involved in the binding of the Fe-S cluster and AdoMet and suggest that tyrosine has contacts near both the C and N termini indicating a folded structure similar to that of BioB (15). This conformation is consistent with the mutant analysis of thiH, particularly the finding that two substitutions (A61S/P334Q and A51V/A373P) are found in some strains with a ThiH* phenotype. In the case of ThiH^A61S/P334Q neither substitution alone disrupted function of the protein (although an A61T substitution was non-functional), yet together they generated a ThiH* phenotype. The similarity of substitutions (A51V/A373P) found in another mutant with a ThiH* phenotype was striking. We interpret these results to indicate that in the folded protein, the N-terminal residues (Ala-52 and Ala-61) are in close proximity with those in the C terminus (Pro-334 and Ala-373). This interpretation is bolstered by the distribution of randomly generated mutations in the primary sequence of ThiH (Fig. 2A). In this analysis, 9/12 residues involved in ThiH* phenotypes and 4/10 causing null mutants are in the first 61 residues or the last 40 residues of the protein.

One interpretation of the apparent intragenic complementation that occurred between different alleles of thiH with a ThiH* phenotype is that ThiH functions in vivo as a homomultimer. Experiments performed in E. coli with overexpressed proteins suggested that the ThiH formed a complex with ThiG (20). This possibility is not excluded by our data. However, comparison with members of the two-component radical AdoMet enzymes, such as pyruvate-formate-lyase activase (PflB) showed that ThiG does not have the characteristic motif RXXGY that provides the glycine loop present in the activation subunit of these enzymes. Additional work, both in vivo and in vitro, is required to clarify the oligomeric state and biochemical mechanism associated with functional ThiH.

	FOOTNOTES

 
^* The competitive grant programs of the National Science Foundation funded this work through Grant MCB0096513. The J. S. McDonnell Foundation provided funds from a 21st Century Scientist Scholars Award. 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.

To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Tel.: 608-265-4630; Fax: 608-265-7909; E-mail: downs{at}bact.wisc.edu.

¹ The abbreviations used are: HMP-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate; THZ-P, thiazole monophosphate; AdoMet, S-adenosylmethionine; HemN, coproporphyrinogen III oxidase.

² P. Frey, personal communication.

³ Available at www.proteinexplorer.org.

	ACKNOWLEDGMENTS

 
We thank Perry Frey for valuable discussion and helpful suggestions. We acknowledge Nicole Baun and Jorge Escalante-Semerena for providing pMETK2 prior to publication.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Begley, T. P., Downs, D. M., Ealick, S. E., McLafferty, F. W., Van Loon, A. P., Taylor, S., Campobasso, N., Chiu, H. J., Kinsland, C., Reddick, J. J., and Xi, J. (1999) Arch. Microbiol. 171, 293–300[CrossRef][Medline] [Order article via Infotrieve]
2. Vander Horn, P. B., Backstrom, A. D., Stewart, V., and Begley, T. P. (1993) J. Bacteriol. 175, 982–992[Abstract/Free Full Text]
3. Lauhon, C. T., and Kambampati, R. (2000) J. Biol. Chem. 275, 20096–20103[Abstract/Free Full Text]
4. 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]
5. 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]
6. Taylor, S. V., Kelleher, N. L., Kinsland, C., Chiu, H. J., Costello, C. A., Backstrom, A. D., McLafferty, F. W., and Begley, T. P. (1998) J. Biol. Chem. 273, 16555–16560[Abstract/Free Full Text]
7. Kambampati, R., and Lauhon, C. T. (2000) J. Biol. Chem. 275, 10727–10730[Abstract/Free Full Text]
8. Sprenger, G. A., Schorken, U., Wiegert, T., Grolle, S., de Graaf, A. A., Taylor, S. V., Begley, T. P., Bringer-Meyer, S., and Sahm, H. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12857–12862[Abstract/Free Full Text]
9. Park, J. H., Dorrestein, P. C., Zhai, H., Kinsland, C., McLafferty, F. W., and Begley, T. P. (2003) Biochemistry 42, 12430–12438[CrossRef][Medline] [Order article via Infotrieve]
10. Leonardi, R., and Roach, P. L. (2004) J. Biol. Chem. 279, 17054–17062[Abstract/Free Full Text]
11. Settembre, E. C., Dorrestein, P. C., Park, J. H., Augustine, A. M., Begley, T. P., and Ealick, S. E. (2003) Biochemistry 42, 2971–2981[CrossRef][Medline] [Order article via Infotrieve]
12. Gralnick, J., Webb, E., Beck, B., and Downs, D. (2000) J. Bacteriol. 182, 5180–5187[Abstract/Free Full Text]
13. 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]
14. Chen, D., Walsby, C., Hoffman, B. M., and Frey, P. A. (2003) J. Am. Chem. Soc. 125, 11788–11789[CrossRef][Medline] [Order article via Infotrieve]
15. Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T., and Drennan, C. L. (2004) Science 303, 76–79[Abstract/Free Full Text]
16. Logan, D. T., Andersson, J., Sjoberg, B. M., and Nordlund, P. (1999) Science 283, 1499–1504[Abstract/Free Full Text]
17. Cosper, M. M., Cosper, N. J., Hong, W., Shokes, J. E., Broderick, W. E., Broderick, J. B., Johnson, M. K., and Scott, R. A. (2003) Protein Sci. 12, 1573–1577[CrossRef][Medline] [Order article via Infotrieve]
18. Skovran, E., and Downs, D. M. (2003) J. Bacteriol. 185, 98–106[Abstract/Free Full Text]
19. Skovran, E., and Downs, D. M. (2000) J. Bacteriol. 182, 3896–3903[Abstract/Free Full Text]
20. Leonardi, R., Fairhurst, S. A., Kriek, M., Lowe, D. J., and Roach, P. L. (2003) FEBS Lett. 539, 95–99[CrossRef][Medline] [Order article via Infotrieve]
21. Miranda-Rios, J., Morera, C., Taboada, H., Davalos, A., Encarnacion, S., Mora, J., and Soberon, M. (1997) J. Bacteriol. 179, 6887–6893[Abstract/Free Full Text]
22. Morett, E., Korbel, J. O., Rajan, E., Saab-Rincon, G., Olvera, L., Olvera, M., Schmidt, S., Snel, B., and Bork, P. (2003) Nat. Biotechnol. 21, 790–795[CrossRef][Medline] [Order article via Infotrieve]
23. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A., and Gelfand, M. S. (2002) J. Biol. Chem. 277, 48949–48959[Abstract/Free Full Text]
24. Vogel, H. J., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97–106[Free Full Text]
25. Petersen, L. A., and Downs, D. M. (1997) J. Bacteriol. 179, 4894–4900[Abstract/Free Full Text]
26. Bartolome, B., Jubete, Y., Martinez, E., and de la Cruz, F. (1991) Gene 102, 75–78[CrossRef][Medline] [Order article via Infotrieve]
27. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410[CrossRef][Medline] [Order article via Infotrieve]
28. Lutz, R., and Bujard, H. (1997) Nucleic Acids Res. 25, 1203–1210[Abstract/Free Full Text]
29. Cosper, N. J., Booker, S. J., Ruzicka, F., Frey, P. A., and Scott, R. A. (2000) Biochemistry 39, 15668–15673[CrossRef][Medline] [Order article via Infotrieve]
30. Way, J. C., Davis, M. A., Morisato, D., Roberts, D. E., and Kleckner, N. (1984) Gene 32, 369–379[CrossRef][Medline] [Order article via Infotrieve]
31. Castilho, B. A., Olfson, P., and Casadaban, M. J. (1984) J. Bacteriol. 158, 488–495[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. M. Boyd, J. L. Sondelski, and D. M. Downs Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function J. Biol. Chem., January 2, 2009; 284(1): 110 - 118. [Abstract] [Full Text] [PDF]

				M. P. Thorgersen and D. M. Downs Oxidative stress and disruption of labile iron generate specific auxotrophic requirements in Salmonella enterica Microbiology, January 1, 2009; 155(1): 295 - 304. [Abstract] [Full Text] [PDF]

				M. Kriek, F. Martins, R. Leonardi, S. A. Fairhurst, D. J. Lowe, and P. L. Roach Thiazole Synthase from Escherichia coli: AN INVESTIGATION OF THE SUBSTRATES AND PURIFIED PROTEINS REQUIRED FOR ACTIVITY IN VITRO J. Biol. Chem., June 15, 2007; 282(24): 17413 - 17423. [Abstract] [Full Text] [PDF]

				M. J. Dougherty and D. M. Downs A connection between iron-sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica. Microbiology, August 1, 2006; 152(Pt 8): 2345 - 2353. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 279/39/40505    most recent M403985200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martinez-Gomez, N. C. Articles by Downs, D. M. Search for Related Content PubMed PubMed Citation Articles by Martinez-Gomez, N. C. Articles by Downs, D. M. Social Bookmarking                      What's this?