scholarly journals SpoT and GppA hydrolases prevent the gratuitous activation of RelA by pppGpp inEscherichia coli

2018 ◽  
Author(s):  
Rajeshree Sanyal ◽  
Rajendran Harinarayanan
Keyword(s):  
Allosteric Regulation ◽  
Growth Arrest ◽  
Stringent Response ◽  
Hydrolase Activity ◽  
Inescherichia Coli ◽  
Modified Nucleotides ◽  
Bistable Switch ◽  
First Time ◽  
Autocatalytic Activation ◽  
Metabolic Cycle

SummaryStringent response, a conserved regulation seen in bacteria, is effected through the modified nucleotides (p)ppGpp. The metabolic cycle of these molecules is driven by the synthase activity of RelA and SpoT and the hydrolase activity of SpoT and GppA which together sets the basal (p)ppGpp pool. Growth arrest due to (p)ppGpp accumulation from basal RelA activity apparently explained the essentiality of SpoT hydrolase function. We found, pppGpp degradation was enhanced when the SpoT hydrolase activity was lowered or eliminated and when this was alleviated by inactivation of the GppA hydrolase, gratuitous synthesis of (p)ppGpp by RelA was activated, leading to growth arrest. The RelA-ribosome interaction was not mandatory for these phenotypes. Our results show, for the first time, elevated pppGpp promoted the amplification of RelA-mediated stringent response in the absence of established RelA activating signals in the cell and the SpoT and GppA hydrolases prevented this. The accumulation of pppGpp inhibited the SpoT hydrolase activity. We propose this autocatalytic activation of RelA by pppGpp is likely to be an allosteric regulation and can result in a bistable switch.

scholarly journals Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift inEscherichia coli

2018 ◽  
Vol 115 (29) ◽  
pp. E6845-E6854 ◽  
Author(s):  
Jae-Woo Lee ◽  
Young-Ha Park ◽  
Yeong-Jae Seok
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Control Mechanism ◽  
Stringent Response ◽  
Hydrolase Activity ◽  
Inescherichia Coli ◽  
Phosphotransferase System ◽  
E Coli ◽  
Cellular Concentration ◽  
Spot Activity

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT inEscherichia coliand related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented inE. coli. In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response inE. coli.

scholarly journals Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition

2020 ◽  
Vol 295 (37) ◽  
pp. 13047-13064 ◽  
Author(s):  
Elfriede Dall ◽  
Florian B. Zauner ◽  
Wai Tuck Soh ◽  
Fatih Demir ◽  
Sven O. Dahms ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Cyclic Peptides ◽  
Molecular Mechanisms ◽  
Functional Studies ◽  
Plant Characteristic ◽  
Activation Mechanisms ◽  
Binding Pockets ◽  
First Time ◽  
Autocatalytic Activation

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGβ is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

scholarly journals A Stringent Analysis of Polyphosphate Dynamics inEscherichia coli

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Michael Downey
Keyword(s):  
Escherichia Coli ◽  
Infection Control ◽  
Bacterial Species ◽  
Stringent Response ◽  
Industrial Applications ◽  
Bacterial Cells ◽  
Inescherichia Coli ◽  
Content Type ◽  
Polyphosphate Accumulation ◽  
Inorganic Phosphates

ABSTRACTDuring stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue, Michael J. Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response inEscherichia coli(M. J. Gray, J. Bacteriol, 201:e00664-18, 2019,https://doi.org/10.1128/JB.00664-18). With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.

scholarly journals Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phasePseudomonas aeruginosa

2018 ◽  
Vol 115 (39) ◽  
pp. 9797-9802 ◽  
Author(s):  
Dorival Martins ◽  
Geoffrey McKay ◽  
Gowthami Sampathkumar ◽  
Malika Khakimova ◽  
Ann M. English ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Stationary Phase ◽  
Growth Arrest ◽  
Stringent Response ◽  
Antioxidant Defenses ◽  
Antibiotic Tolerance ◽  
Sod Activity ◽  
Downstream Effectors ◽  
Key Factor ◽  
Multidrug Tolerance

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses inPseudomonas aeruginosa. We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phaseP. aeruginosa. Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of theΔrelA spoTmutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phaseP. aeruginosaand suggest that inhibition of SOD activity may potentiate current antibiotics.

The stringent response to inhibition of peptide chain initiation inEscherichia coli

FEBS Letters ◽  
1976 ◽  
Vol 71 (1) ◽  
pp. 103-106 ◽  
Author(s):  
J. Roche ◽  
Y. Cenatiempo ◽  
A.J. Cozzone
Keyword(s):  
Stringent Response ◽  
Peptide Chain ◽  
Inescherichia Coli ◽  
Chain Initiation

A dimeric catalytic core relates the short and long forms of ATP-phosphoribosyltransferase

2018 ◽  
Vol 475 (1) ◽  
pp. 247-260 ◽  
Author(s):  
Gerd Mittelstädt ◽  
Wanting Jiao ◽  
Emma K. Livingstone ◽  
Gert-Jan Moggré ◽  
Ali Reza Nazmi ◽  
...  
Keyword(s):  
Short Form ◽  
Allosteric Regulation ◽  
Regulatory Domain ◽  
Molecular Architecture ◽  
Histidine Biosynthesis ◽  
Catalytic Core ◽  
Catalytic Domains ◽  
Long Form ◽  
Covalently Linked ◽  
First Time

Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an allosteric inhibitor. Here, we describe a truncated long-form ATP-PRT from Campylobacter jejuni devoid of its regulatory domain (CjeATP-PRTcore). Results showed that CjeATP-PRTcore is dimeric, exhibits attenuated catalytic activity, and is insensitive to histidine, indicating that the covalently linked regulatory domain plays a role in both catalysis and regulation. Crystal structures were obtained for CjeATP-PRTcore in complex with both substrates, and for the first time, the complete product of the reaction. These structures reveal the key features of the active site and provide insights into how substrates move into position during catalysis.

scholarly journals Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: Insights from accelerated molecular dynamics simulations

2019 ◽  
Author(s):  
Juan A. Bueren-Calabuig ◽  
Marcus Bage ◽  
Victoria H. Cowling ◽  
Andrei V. Pisliakov
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Allosteric Regulation ◽  
Dynamic Network ◽  
Binding Model ◽  
Accelerated Molecular Dynamics ◽  
Dynamics Simulations ◽  
First Time

ABSTRACTThe RNA guanine-7 methyltransferase (RNMT) in complex with RNMT-Activating Miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5’ end of nascent RNA polymerase II transcripts. The mRNA cap protects the transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the dynamic network and community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

scholarly journals Modified nucleotides inBacillus subtilistRNATrphyperexpressed inEscherichia coli

1993 ◽  
Vol 21 (10) ◽  
pp. 2479-2486 ◽  
Author(s):  
Hong Xue ◽  
Anne-Lise Glasser ◽  
Jean Desgres ◽  
Henri Grosjean
Keyword(s):  
Inescherichia Coli ◽  
Modified Nucleotides

scholarly journals The stringent response in Myxococcus xanthus is regulated by SocE and the CsgA C-signaling protein

2000 ◽  
Vol 14 (4) ◽  
pp. 483-492 ◽  
Author(s):  
Eugene W. Crawford ◽  
Lawrence J. Shimkets
Keyword(s):  
Amino Acid ◽  
Myxococcus Xanthus ◽  
Growth Arrest ◽  
Stringent Response ◽  
Binding Pocket ◽  
Macromolecular Synthesis ◽  
New Members ◽  
Coenzyme Binding ◽  
Body Development ◽  
Fruiting Body Development

Myxococcus xanthus fruiting body development is induced by amino acid limitation. The decision to grow or develop is established by the RelA-dependent stringent response and A-signaling. We identified two new members of this regulatory hierarchy, socE and the C-signaling gene csgA. SocE depletion arrests growth and induces sporulation under conditions that normally favor growth as well as curtailing DNA and stable RNA synthesis, inhibiting cell elongation, and inducing accumulations of the stringent nucleotides ppGpp and pppGpp [(p)ppGpp]. This system separates C-signaling, which does not occur under these conditions, from CsgA enzyme activity. Amino acid substitutions in the CsgA coenzyme binding pocket or catalytic site eliminate growth arrest. relA mutation also eliminates growth arrest. Eleven pseudorevertants selected for growth following SocE depletion contained mutations in csgA or relA. These results suggest that CsgA induces the stringent response and while SocE inhibits it. Unlike the csgA mutant, wild-type and socE csgA cells maintained high levels of (p)ppGpp throughout development. We suggest that CsgA maintains growth arrest throughout development to divert carbon from A-signaling and other sources into developmental macromolecular synthesis.

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