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Bacteria have a remarkable ability to sense environmental stresses and to respond to these stressors by adapting their metabolism and physiology. In recent publications, investigators have suggested that multiple stresses that cause cell death share the mechanistic feature of stimulating the formation of reactive oxygen species (ROS). A central piece of evidence cited in these claims is the ability of exogenous antioxidant compounds to mitigate stress‐related cell death. The validity of attributing a positive effect of exogenous antioxidants to ROS‐mediated stress is challenged by an important study by Korshunov and Imlay in this issue of Molecular Microbiology. This study reports biochemical data that convincingly show that some commonly used antioxidants quench oxidants orders of magnitude too slowly to have a significant effect on the concentration of ROS in the cell. Under conditions where antioxidants minimize cell death, they also slow growth. Significantly, slowing cell growth by other means has the same restorative effect as adding an antioxidant. Based on the solid biochemical and genetic data, Korshunov and Imlay make the case for discarding the use of antioxidants to diagnose conditions that generate increased internal ROS production. [ABSTRACT FROM AUTHOR]

The RidA subfamily of the Rid (YjgF/YER057c/UK114) superfamily of proteins is broadly distributed and found in all domains of life. RidA proteins are enamine/imine deaminases. In the organisms that have been investigated, lack of RidA results in accumulation of the reactive enamine species 2‐aminoacrylate (2AA) and/or its derivative imine 2‐iminopropanoate (2IP). The accumulated enamine/imine species can damage specific pyridoxal phosphate (PLP)‐dependent target enzymes. The metabolic imbalance resulting from the damaged enzymes is organism specific and based on metabolic network configuration. Saccharomyces cerevisiae encodes two RidA homologs, one localized to the cytosol and one to the mitochondria. The mitochondrial RidA homolog, Mmf1p, prevents enamine/imine stress and is important for normal growth and maintenance of mitochondrial DNA. Here, we show that Mmf1p is necessary for optimal heme biosynthesis. Biochemical and/or genetic data herein support a model in which accumulation of 2AA and or 2IP, in the absence of Mmf1p, inactivates Hem1p, a mitochondrially located PLP‐dependent enzyme required for heme biosynthesis. [ABSTRACT FROM AUTHOR]

YggS is a pyridoxal 5′‐phosphate (PLP)‐binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP‐dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP‐dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5′‐phosphate and α‐keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination. [ABSTRACT FROM AUTHOR]

The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage of LpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5. Importance: Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function. [ABSTRACT FROM AUTHOR]

Summary: The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5′‐phosphate (PLP)‐binding proteins that play a critical role in the homeostasis of vitamin B6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3‐phosphoglycerate dehydrogenase) were conditionally lethality in the M9‐glucose medium supplemented with Gly. Analyses of vitamin B6 pools found the high‐levels of pyridoxine 5′‐phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP‐dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants. [ABSTRACT FROM AUTHOR]

Building a robust, stable network must include strategies to minimize perturbations caused by environmental stress, while optimizing cellular fitness. The introduction of oxygen into the Earth's atmosphere brought challenges for the microbes that had evolved enzyme machinery and metabolic network stability in the anoxic world. Unable to generate new enzyme paradigms and metabolic networks de novo, organisms have evolved strategies to neutralize the impact of oxygen that can be added to and integrated into the existing metabolic framework. This issue of Molecular Microbiology includes a paper by Korshunov et al. in which the authors describe an elegant strategy that Escherichia coli has evolved to minimize metabolic stress that results from the acquisition and use of cystine, the oxidized form of cysteine, as a source of cellular sulfur. This study highlights how a strategy involving both cost and benefit can result in a functional, but energy intensive mechanism for this bacterium to thrive in an oxic world. [ABSTRACT FROM AUTHOR]

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L-2,3-diaminopropionate (Dap) is a non-protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia-lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL-dependent degradation of Dap to pyruvate proceeds through an unbound 2-aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaLdependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification. [ABSTRACT FROM AUTHOR]

The Rid family of proteins is highly conserved and broadly distributed throughout the domains of life. Genetic and biochemical studies, primarily in S almonella enterica, have defined a role for RidA in responding to endogenously generated reactive metabolites. The data show that 2-aminoacrylate (2 AA), a reactive enamine intermediate generated by some pyridoxal 5′-phosphate-dependent enzymes, accumulates in the absence of RidA. The accumulation of 2 AA leads to covalent modification and inactivation of several enzymes involved in essential metabolic processes. This review describes the 2 AA hydrolyzing activity of RidA and the effect of this biochemical activity on the metabolic network, which impacts organism fitness. The reported activity of RidA and the consequences encountered in vivo when RidA is absent have challenged fundamental assumptions in enzymology, biochemistry and cell metabolism regarding the fate of transiently generated reactive enamine intermediates. The current understanding of RidA in S almonella and the broad distribution of Rid family proteins provide exciting opportunities for future studies to define metabolic roles of Rid family members from microbes to man. [ABSTRACT FROM AUTHOR]

The RidA/ Yer057/ UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2- AA) accumulates and inactivates at least some pyridoxal 5′-phosphate ( PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2- AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild-type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase ( GlyA; E. C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella. [ABSTRACT FROM AUTHOR]

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a β1β2α1α2α3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the 1H-15N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought. Proteins 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]