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Bacterial quorum sensing (QS) is a cell-cell communication process, mediated by signaling molecules, that alters various phenotypes including pathogenicity. Methods to interrupt these communication networks are being pursued as next generation antimicrobials. We present a technique for interrupting communication among bacteria that exploits their native and highly specific machinery for processing the signaling molecules themselves. Specifically, our approach is to bring native intracellular signal processing mechanisms to the extracellular surroundings and "quench" crosstalk among a variety of strains. In this study, the QS system based on the interspecies signaling molecule autoinducer-2 (AI-2) is targeted because of its prevalence among prokaryotes (it functions in over 80 bacterial species). We demonstrate that the Escherichia coli AI-2 kinase, LsrK, can phosphorylate AI-2 in vitro, and when LsrK-treated AI-2 is added ex vivo to E. coli populations, the native QS response is significantly reduced. Further, LsrK-mediated degradation of AI-2 attenuates the QS response among Salmonella typhimurium and Vibrio harveyi even though the AI-2 signal transduction mechanisms and the phenotypic responses are species-specific. Analogous results are obtained from a synthetic ecosystem where three species of bacteria (enteric and marine) are co-cultured. Finally, the addition of LsrK and ATP to growing co-cultures of E. coli and S. typhimurium exhibits significantly reduced native "cross-talk" that ordinarily exists among and between species in an ecosystem. We believe this nature-inspired enzymatic approach for quenching QS systems will spawn new methods for controlling cell phenotype and potentially open new avenues for controlling bacterial pathogenicity.
ACS Chem Biol 2010 Feb 19
PMID:Cross species quorum quenching using a native AI-2 processing enzyme. 2002 44

Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation.
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PMID:Acetate formation in the energy metabolism of parasitic helminths and protists. 2008 67

Fluorescence assays for ADP detection are of considerable current interest, both in basic research and in drug discovery, as they provide a generic method for measuring the activity of ATPases and kinases. The development of a novel fluorescent biosensor is described that is based on a tetramethylrhodamine-labeled, bacterial actin homologue, ParM. The design of the biosensor takes advantage of the large conformational change of ParM on ADP binding and the strong quenching of the tetramethylrhodamine fluorescence by stacking of the dye. ParM was labeled with two tetramethylrhodamines in close proximity, whereby the fluorophores are able to interact with each other. ADP binding alters the distance and relative orientation of the tetramethylrhodamines, which leads to a change in this stacking interaction and so in the fluorescence intensity. The final ADP biosensor shows approximately 15-fold fluorescence increase in response to ADP binding. It has relatively weak affinity for ADP (K(d) = 30 microM), enabling it to be used at substoichiometric concentrations relative to ADP, while reporting ADP concentration changes in a wide range around the K(d) value, namely, submicromolar to tens of micromolar. The biosensor strongly discriminates against ATP (>100-fold), allowing ADP detection against a background of millimolar ATP. At 20 degrees C, the labeled ParM binds ADP with a rate constant of 9.5 x 10(4) M(-1) s(-1) and the complex dissociates at 2.9 s(-1). Thus, the biosensor is suitable for real-time measurements, and its performance in such assays is demonstrated using a sugar kinase and a mammalian protein kinase.
ACS Chem Biol 2010 Apr 16
PMID:A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM. 2015 67

Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.
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PMID:SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. 2020 11

Here we use the fluorescence from a genetically encoded unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly), replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the molecular details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissociation between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asymmetric allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concentrations.
ACS Chem Biol 2010 May 21
PMID:Asymmetric allosteric signaling in aspartate transcarbamoylase. 2021 Mar 58

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.
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PMID:Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation. 2023 19

The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg(2+)-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0-2.8 A. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nepsilon(2) nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.
ACS Chem Biol 2010 Jun 18
PMID:Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates. 2039 12

A simple ADP biosensor would be of broad usefulness in monitoring the large number of metabolic processes that produce ADP. Several new systems have been recently described including one in the current issue of ACS Chemical Biology that provides a simple readout of the ADP concentration without significant interference by ATP.
ACS Chem Biol 2010 Apr 16
PMID:When is weaker better? Design of an ADP sensor with weak ADP affinity, but still selective against ATP. 2039 42

Isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. They are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In Archaea and Eukarya, these building blocks are synthesized via the mevalonate pathway. However, the genes required to convert mevalonate phosphate (MP) to IPP are missing in several species of Archaea. An enzyme with isopentenyl phosphate kinase (IPK) activity was recently discovered in Methanocaldococcus jannaschii (MJ), suggesting a departure from the classical sequence of converting MP to IPP. We have determined the high-resolution crystal structures of isopentenyl phosphate kinases in complex with both substrates and products from Thermoplasma acidophilum (THA), as well as the IPK from Methanothermobacter thermautotrophicus (MTH), by means of single-wavelength anomalous diffraction (SAD) and molecular replacement. A histidine residue (His50) in THA IPK makes a hydrogen bond with the terminal phosphates of IP and IPP, poising these molecules for phosphoryl transfer through an in-line geometry. Moreover, a lysine residue (Lys14) makes hydrogen bonds with nonbridging oxygen atoms at P(alpha) and P(gamma) and with the P(beta)-P(gamma) bridging oxygen atom in ATP. These interactions suggest a transition-state-stabilizing role for this residue. Lys14 is a part of a newly discovered "lysine triangle" catalytic motif in IPKs that also includes Lys5 and Lys205. Moreover, His50, Lys5, Lys14, and Lys205 are conserved in all IPKs and can therefore serve as fingerprints for identifying new homologues.
ACS Chem Biol 2010 May 21
PMID:X-ray structures of isopentenyl phosphate kinase. 2040 38

Pancreatic beta-cell apoptosis is a critical event during the development of type-1 diabetes. The identification of small molecules capable of preventing cytokine-induced apoptosis could lead to avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays designed to identify such small-molecule suppressors. Rat INS-1E cells were simultaneously treated with a cocktail of inflammatory cytokines and a collection of 2,240 diverse small molecules and screened using an assay for cellular ATP levels. Forty-nine top-scoring compounds included glucocorticoids, several pyrazole derivatives, and known inhibitors of glycogen synthase kinase-3beta. Two compounds were able to increase cellular ATP levels, reduce caspase-3 activity and nitrite production, and increase glucose-stimulated insulin secretion in the presence of cytokines. These results indicate that small molecules identified by this screening approach may protect beta cells from autoimmune attack and may be good candidates for therapeutic intervention in early stages of type-1 diabetes.
ACS Chem Biol 2010 Aug 20
PMID:Small-Molecule Suppressors of Cytokine-Induced beta-Cell Apoptosis. 2055 Jan 76


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