Metabolic Defects Caused by Mutations in the
            <i>isc</i>
            Gene Cluster in
            <i>Salmonella enterica</i>
            Serovar Typhimurium: Implications for Thiamine Synthesis

Skovran, Elizabeth; Downs, Diana M.

doi:10.1128/jb.182.14.3896-3903.2000

Cited by 68 publications

(98 citation statements)

References 60 publications

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“…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.…”

Section: Strains-mentioning

confidence: 99%

“…Cell pellets from 3 ml of culture were frozen at Ϫ20°C and assayed within 1 week. Pellets were resuspended and washed in 300 l of 20 mM Tris citrate buffer (pH 8) and assayed as described previously (12).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Lauhon

Skovran

Urbina

et al. 2004

Journal of Biological Chemistry

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IscS catalyzes the fragmentation of L-cysteine to Lalanine 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 Cys 328 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 Cys 328 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 (s 2 C and ms 2 i 6 A) yet showed wild type levels of Fe-S-independent thionucleosides (s 4 U and mnm 5 s 2 U) 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.

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Section: Strains-mentioning

confidence: 99%

Section: Enzyme Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

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

Lauhon

Skovran

Urbina

et al. 2004

Journal of Biological Chemistry

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“…The induction of metK in plasmid pMETK2 increases AdoMet synthase activity Ͼ5-fold. 2 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).…”

Section: Effect Of Mutations Affecting the Putative [Fe-s] Clustermentioning

confidence: 96%

“…The characterized proteins of this family generate radical species by reductive cleavage of AdoMet at the [Fe-S] cluster, and structural and biochemical data have shown that the AdoMet molecule binds either the iron that is not liganded by the protein or one of the 3 -bridging sulfides of the cluster (14 -17). Evidence for the presence of an [Fe-S] cluster in ThiH has been provided by genetic (18,19) and biochemical (20) studies. However, data directly tying the [Fe-S] cluster to enzymatic activity have not been reported.…”

mentioning

confidence: 99%

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

Martinez‐Gomez

Robers

Downs

2004

Journal of Biological Chemistry

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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.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 5Ј-deoxyadenosyl radical, generating a tryosyl radical that results in the glycine imine via a series of steps (10).Genetic studies identified a connection between thiamine biosynthesis and [Fe-S] cluster metabolism in Salmonella enterica and suggested that ThiH was the weak link in thiazole synthesis (12). ThiH is a 45-kDa protein that was identified as a member of the radical AdoMet superfamily of proteins (13). Proteins in this superfamily are characte...

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Iron–Sulfur Cluster Biogenesis in Archaea and Bacteria

Chahal

Boyd

Outten

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Iron–sulfur (Fe–S) clusters are key cofactors required for the activity of essential metalloproteins. Biogenesis of Fe–S clusters is carried out by multiple proteins that work together to mobilize iron and sulfide, assemble nascent clusters, and traffic them to target metalloproteins. While the Isc, Nif, and Suf pathways are the most well‐studied Fe–S cluster biogenesis systems, it is clear that a variety of accessory proteins and metabolites cooperate with these systems for cluster assembly. Here, we review the current status of Fe–S cluster biogenesis research in Bacteria and Archaea.

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Metabolic Defects Caused by Mutations in the isc Gene Cluster in Salmonella enterica Serovar Typhimurium: Implications for Thiamine Synthesis

Cited by 68 publications

References 60 publications

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

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

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

Iron–Sulfur Cluster Biogenesis in Archaea and Bacteria

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