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

Palmer, Lauren D.; Dougherty, Michael J.; Downs, Diana M.

doi:10.1128/jb.01361-12

Cited by 9 publications

(12 citation statements)

References 44 publications

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“…The ability of these metabolites to suppress the growth defect independently may reflect a one-carbon sparing phenomenon. Alternatively, because coenzyme A and methionine are known to stimulate production of thiamine in S. enterica (25), these data may indicate that a thiamine limitation is the key to the growth defect observed for the ilvA219 ridA strain.…”

Section: Resultsmentioning

confidence: 99%

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

Ernst

Downs

2016

J Bacteriol

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The reactive enamine 2-aminoacrylate (2AA) is a metabolic stressor capable of damaging cellular components. Members of the broadly conserved Rid (RidA/YER057c/UK114) protein family mitigate 2AA stress in vivo by facilitating enamine and/or imine hydrolysis. Previous work showed that 2AA accumulation in ridA strains of Salmonella enterica led to the inactivation of multiple target enzymes, including serine hydroxymethyltransferase (GlyA). However, the specific cause of a ridA strain's inability to grow during periods of 2AA stress had yet to be determined. Work presented here shows that glycine supplementation suppressed all 2AA-dependent ridA strain growth defects described to date. Depending on the metabolic context, glycine appeared to suppress ridA strain growth defects by eliciting a GcvB small RNA-dependent regulatory response or by serving as a precursor to one-carbon units produced by the glycine cleavage complex (GCV). In either case, the data suggest that GlyA is the most physiologically sensitive target of 2AA inactivation in S. enterica. The universally conserved nature of GlyA among free-living organisms highlights the importance of RidA in mitigating 2AA stress. IMPORTANCEThe RidA stress response prevents 2-aminoacrylate (2AA) damage from occurring in prokaryotes and eukaryotes alike. 2AA inactivation of serine hydroxymethyltransferase (GlyA) from Salmonella enterica restricts glycine and one-carbon production, ultimately reducing fitness of the organism. The cooccurrence of genes encoding 2AA production enzymes and serine hydroxymethyltransferase (SHMT) in many genomes may in part underlie the evolutionary selection for Rid proteins to maintain appropriate glycine and one-carbon metabolism throughout life. R id (RidA/YER057c/UK114) proteins are found throughout each kingdom of life, indicating that these proteins play fundamental roles in cellular processes. Bioinformatics analyses identified eight subgroups within the Rid protein family (1). The archetypal RidA subfamily is broadly distributed and includes the best-studied RidA enzyme from Salmonella enterica, while the remaining seven Rid subgroups are primarily restricted to bacteria. In S. enterica, RidA is known to preempt metabolic damage caused by the unbound reactive enamine 2-aminoacrylate (2AA) (2). In the absence of RidA, 2AA persisted in solution long enough to inactivate multiple target enzymes in vivo and in vitro (2-4). RidA proteins isolated from organisms spanning the tree of life displayed similar enamine and imine deaminase activities (5, 6), suggesting that RidA enzymes have a conserved role in preventing 2AA stress.2AA stress arises as a consequence of some pyridoxal 5=-phosphate (PLP)-dependent enzyme activities. Previous analysis of S. enterica showed that PLP-dependent serine/threonine dehydratases (IlvA/TdcB; EC 4.3.1.19), cysteine desulfhydrase (CdsH; EC 4.4.1.1), and diaminopropionate ammonia-lyase (DpaL; EC 4.3.1.15) generated free 2AA from exogenous serine, cysteine, and diaminopropionate, respectively, leading ...

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Section: Resultsmentioning

confidence: 99%

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

Ernst

Downs

2016

J Bacteriol

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“…1.18.1.2), flavodoxin A (FldA), and TdPurE were expressed and purified as described previously (26,30). TdPurE is Treponema denticola AIR carboxylase (E.C.…”

Section: Methodsmentioning

confidence: 99%

“…S. enterica His 6 -ThiC was produced from vector pET-28b(ϩ) in a strain overexpressing Azotobacter vinelandii [Fe-S] cluster-loading genes from plasmid pDB1282 (31). ThiC was purified as described (26), except that the [4Fe-4S] cluster was reconstituted in vitro prior to freezing the protein at Ϫ80°C. After purification, ThiC concentration was determined by Pierce 660 assay (Thermo Scientific, Rockford, IL) using BSA as the standard.…”

Section: Methodsmentioning

confidence: 99%

The Thiamine Biosynthetic Enzyme ThiC Catalyzes Multiple Turnovers and Is Inhibited by S-Adenosylmethionine (AdoMet) Metabolites

Palmer

Downs

2013

Journal of Biological Chemistry

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Background: ThiC is a radical S-adenosylmethionine (AdoMet) enzyme that synthesizes the thiamine pyrimidine 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P). Results: An increase in the ThiC catalytic rate was detected when product 5Ј-deoxyadenosine was hydrolyzed. ThiC was inhibited by AdoMet metabolites. Conclusion: ThiC is a multiple-turnover enzyme and is product-inhibited. Significance: This is the first report of ThiC catalytic turnover and the identification of two AdoMet metabolites that inhibit ThiC activity.

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“…The plates were incubated at 37°C for 18 h and assessed for growth. ThiC assay components (ThiC, authentic AIR, and purified SAM) were a gift from L. D. Palmer and were prepared as previously described (19,31). Previous experiments determined that a bioassay with DM7185 reliably detected 1 pmol of HMP (L. D. Palmer, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

“…For conditions that favored reverse catalysis, the final reaction volumes were 50 l and contained the following: 50 mM Tris, 20 mM KCl, 6 mM MgCl 2 , 2.5 mM Na 3 AsO 4 , 2.5 mM ADP, 2.5 mM AICAR, 100 M PurC, and 4 M TdPurE. TdPurE was a gift from L. D. Palmer (31). TdPurE, which can interconvert AIR and CAIR, was added in case AIR synthesis proceeded through a CAIR intermediate.…”

Section: Methodsmentioning

confidence: 99%

Aminoimidazole Carboxamide Ribotide Exerts Opposing Effects on Thiamine Synthesis in Salmonella enterica

2015

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In Salmonella enterica, the thiamine biosynthetic intermediate 5-aminoimidazole ribotide (AIR) can be synthesized de novo independently of the early purine biosynthetic reactions. This secondary route to AIR synthesis is dependent on (i) 5-amino-4-imidazolecarboxamide ribotide (AICAR) accumulation, (ii) a functional phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase (PurC; EC 6.3.2.6), and (iii) methionine and lysine in the growth medium. Studies presented here show that AICAR is a direct precursor to AIR in vivo. PurC-dependent conversion of AICAR to AIR was recreated in vitro. Physiological studies showed that exogenous nutrients (e.g., methionine and lysine) antagonize the inhibitory effects of AICAR on the ThiC reaction and decreased the cellular thiamine requirement. Finally, genetic results identified multiple loci that impacted the effect of AICAR on thiamine synthesis and implicated cellular aspartate levels in AICAR-dependent AIR synthesis. Together, the data here clarify the mechanism that allows conditional growth of a strain lacking the first five biosynthetic enzymes, and they provide additional insights into the complexity of the metabolic network and its plasticity. IMPORTANCEIn bacteria, the pyrimidine moiety of thiamine is derived from aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. A previous study described conditions under which AIR synthesis is independent of purine biosynthesis. This work is an extension of that previous study and describes a new synthetic pathway to thiamine that depends on a novel thiamine precursor and a secondary activity of the biosynthetic enzyme PurC. These findings provide mechanistic details of redundancy in the synthesis of a metabolite that is essential for nucleotide and coenzyme biosynthesis. Metabolic modifications that allow the new pathway to function or enhance it are also described.

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

Cited by 9 publications

References 44 publications

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica

The Thiamine Biosynthetic Enzyme ThiC Catalyzes Multiple Turnovers and Is Inhibited by S-Adenosylmethionine (AdoMet) Metabolites

Aminoimidazole Carboxamide Ribotide Exerts Opposing Effects on Thiamine Synthesis in Salmonella enterica

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