A connection between iron–sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica

Dougherty, Michael J.; Downs, Diana M.

doi:10.1099/mic.0.28926-0

Cited by 30 publications

(37 citation statements)

References 39 publications

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“…Since deficiency of the branched-chain amino acids (BCAA) [49] and thiamin [50] has been considered a hallmark of oxidative inactivation of iron-sulfur enzymes in bacteria, we postulated that deletion of both IscA and SufA may prevent the iron-sulfur cluster assembly in key enzymes, thus blocking the biosyntheses of BCAA and thiamin in E. coli cells. Figure 1C shows that addition of the three branched-chain amino acids (leucine, isoleucine and valine) indeed partially restored cell growth of the E. coli iscA/sufA double mutant in minimal medium under aerobic growth conditions.…”

Section: Resultsmentioning

confidence: 99%

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

Tan

Lü

Bitoun

et al. 2009

Biochemical Journal

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SynopsisIscA/SufA paralogs are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. While deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/ SufA paralogs blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthesis pathway in E. coli cells under aerobic conditions and that addition of the ironbound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the cluster assembly in protein ThiC of the thiamin biosynthesis pathway, aconitase B of the citrate acid cycle, and endonuclease III of the DNA base excision repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin, and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogs are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions. Keywords aconitase; branched-chain amino acids; dihydroxyacid dehydratase; iron-sulfur clusters; IscA/SufA paralogs; thiamin IntroductionIron-sulfur clusters are one of the most ancient and ubiquitous redox centers in biology. They are involved in diverse physiological processes including respiratory electron transfer, nitrogen fixation, photosynthesis, biosynthesis of amino acids, thiamin, heme, biotin, and lipoic acid, DNA synthesis and repair, RNA modification, and the regulation of gene expression [1,2]. However, the mechanism underlying the iron-sulfur cluster assembly is still not fully understood [3]. The discovery of cysteine desulfurase (NifS) Experimental Mutant strains and cell growthThe E. coli deletion mutants in which iscA and sufA were in-frame deleted were previously constructed [28]. Each deletion in E. coli cells was confirmed by PCR as described in [28]. For cell growth analysis, overnight cell cultures grown in Luria-Bertani (LB) medium were washed twice with minimal medium containing glucose (0.2%) before inoculated on minimal medium plates or in liquid minimal medium at 37°C with aeration (250 rpm). Cell growth was recorded by measuring the optical density of cell culture at 600 nm. When indicated, minimal medium was supplemented with the three branched-chain amino acids ...

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Section: Resultsmentioning

confidence: 99%

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

Tan

Lü

Bitoun

et al. 2009

Biochemical Journal

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“…Instead, ThiC contains a CXXCXXXXC motif. The indispensability of this motif was confirmed by a mutational study showing that replacement of any cysteine residue with alanine abolished thiamine biosynthesis (29). Modeling a [4Fe-4S] cluster in the apo-ThiC crystal structure guided by a biotin synthase backbone supported the function of the CXXCXXXXC motif for binding a [4Fe-4S] cluster (16), which has been shown to exist in the reconstituted protein by EPR, UV-visible, and Mössbauer spectrometries (20,21).…”

Section: Hmp-p Synthase Thic In Thiamine Biosynthesismentioning

confidence: 84%

Complex Biotransformations Catalyzed by Radical S-Adenosylmethionine Enzymes

Zhang

2011

Journal of Biological Chemistry

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The radical S-adenosylmethionine (AdoMet) superfamily currently comprises thousands of proteins that participate in numerous biochemical processes across all kingdoms of life. These proteins share a common mechanism to generate a powerful 5-deoxyadenosyl radical, which initiates a highly diverse array of biotransformations. Recent studies are beginning to reveal the role of radical AdoMet proteins in the catalysis of highly complex and chemically unusual transformations, e.g. the ThiC-catalyzed complex rearrangement reaction. The unique features and intriguing chemistries of these proteins thus demonstrate the remarkable versatility and sophistication of radical enzymology.

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“…Residues that are substituted in variants identified in this study are highlighted on the basis of the phenotype that their presence causes in vivo: null phenotype (black) and conditional thiamine auxotrophs (gray). Asterisks above the residues indicate that their null phenotype was characterized by site-directed mutagenesis in a previous study (13). growth conditions that had predictable consequences for the metabolic network.…”

Section: Resultsmentioning

confidence: 99%

“…In vivo experiments found that compromised iron-sulfur ([Fe-S]) cluster metabolism reduced ThiC activity, results that contributed to the identification of the [Fe-S] requirement of this protein (13). In other studies, strains with reduced coenzyme A (CoA) levels and those that accumulated the purine intermediate 5-aminoimidazole-4-carboxamide ribotide (AICAR) have less ThiC-dependent growth in vivo (1,20).…”

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confidence: 99%

Analysis of ThiC Variants in the Context of the Metabolic Network of Salmonella enterica

2012

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In bacteria, the 4-amino-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine is synthesized from 5-aminoimidazole ribotide (AIR), a branch point metabolite of purine and thiamine biosynthesis. ThiC is a member of the radical S-adenosylmethionine (AdoMet) superfamily and catalyzes the complex chemical rearrangement of AIR to HMP-P. As reconstituted in vitro, the ThiC reaction requires AdoMet, AIR, and reductant. This study analyzed variants of ThiC in vivo and in vitro to probe the metabolic network surrounding AIR in Salmonella enterica. Several variants of ThiC that required metabolic perturbations to function in vivo were biochemically characterized in vitro. Results presented herein indicate that the subtleties of the metabolic network have not been captured in the current reconstitution of the ThiC reaction.

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A connection between iron–sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica

Cited by 30 publications

References 39 publications

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in enzymes of multiple physiological pathways inEscherichia coliunder aerobic growth conditions

Complex Biotransformations Catalyzed by Radical S-Adenosylmethionine Enzymes

Analysis of ThiC Variants in the Context of the Metabolic Network of Salmonella enterica

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