The Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate

Cabrera, Ricardo; Báez, María E.; Pereira, H.M.; Caniuguir, A.; Garratt, R.C.; Babul, Jorge

doi:10.1074/jbc.m110.163162

Cited by 28 publications

(36 citation statements)

References 37 publications

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“…The binding of the substrate molecules to all (or most) of the 8 active sites in an octamer causes the structural change of each subunit, e.g., winding of the N-terminal ␣-helix, and then leads to dissociation into tetramers, which can catalyze isomerization of the substrate. The change in the quaternary structure of the enzyme is reminiscent of that of a well-studied allosteric enzyme, phosphofructokinase-2 (PFK-2) from E. coli (3)(4)(5). The E. coli enzyme takes inactive homotetrameric configuration in the presence of a homotrophic inhibitor, MgATP, which binds not only to the active sites but also to the effector sites, but turns into active homodimers when fructose-6-phosphate binds to the active sites.…”

Section: Discussionmentioning

confidence: 99%

Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

et al. 2012

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Type 2 isopentenyl diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylallyl diphosphate, in almost all archaea and in some bacteria, including human pathogens. The enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 isopentenyl diphosphate isomerase. However, type 2 enzymes were reportedly inhibited by mechanism-based drugs for the type 1 enzyme due to their surprisingly similar reaction mechanisms. Thus, a different approach is now required to develop new inhibitors specific to the type 2 enzyme. X-ray crystallography and gel filtration chromatography revealed that the enzyme from a thermoacidophilic archaeon, Sulfolobus shibatae , is in the octameric state at a high concentration. Interestingly, a part of the regions that are involved in the substrate binding in the previously reported tetrameric structures is integral to the formation of the tetramer-tetramer interface in the substrate-free octameric structure. Site-directed mutagenesis at such regions resulted in stabilization of the tetramer. Small-angle X-ray scattering, tryptophan fluorescence, and dynamic light scattering analyses showed that substrate binding causes the dissociation of an octamer into tetramers. This property, i.e., incompatibility between octamer formation and substrate binding, might provide clues to develop new specific inhibitors of the archaeal enzyme.

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Section: Discussionmentioning

confidence: 99%

Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

et al. 2012

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“…We included 17 allosteric regulation terms, taken from literature, in the CFPS model. PEP was modeled as an inhibitor for phosphofructokinase [29, 30], PEP carboxykinase [29], PEP synthetase [29, 31], isocitrate dehydrogenase [29, 32], and isocitrate lyase/malate synthase [29, 32, 33], and as an activator for fructose-biphosphatase [29, 34, 35, 36]. AKG was modeled as an inhibitor for citrate synthase [29, 37, 38] and isocitrate lyase/malate synthase [29, 33].…”

Section: Methodsmentioning

confidence: 99%

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis inEscherichia coli

Horvath

Vilkhovoy²,

Wayman

et al. 2017

Preprint

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Cell-free protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamic E. coli cell-free protein synthesis (CFPS) models. Model parameters were estimated from a training dataset for the cell-free production of a protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluconeogenesis. Amino acid biosynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and should provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.Introduction 1 Cell-free protein expression has become a widely used research tool in 2 systems and synthetic biology, and a promising technology for personalized 3 point of use biotechnology [1]. Cell-free systems offer many advantages for 4 the study, manipulation and modeling of metabolism compared to in vivo 5 processes. Central amongst these, is direct access to metabolites and the 6 biosynthetic machinery without the interference of a cell wall, or complica-7 tions associated with cell growth. This allows us to interrogate (and po-8 tentially manipulate) the chemical microenvironment while the biosynthetic 9 machinery is operating, potentially at a fine time resolution. Cell-free pro-10 tein synthesis (CFPS) systems are arguably the most prominent examples 11 of cell-free systems used today [2]. However, CFPS is not new; CFPS in 12 crude E. coli extracts has been used since the 1960s to explore fundamental 13 biological mechanisms. For example, Matthaei and Nirenberg used E. coli 14 cell-free extract in ground-breaking experiments to decipher the sequencing 15 of the genetic code [3, 4]. Spirin and coworkers later improved protein pro-16 duction in cell free extracts by continuously exchanging reactants and prod-17 ucts; however, while these extracts could run for tens of hours, they could 18 only synthesize a single product and were energy limited [5]. More recently, 19 energy and cofactor regeneration in CFPS has been significantly improved; 20 for example ATP can be regenerated using substrate level pho...

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“…In contrast, at physiological conditions favoring the monomeric form of the enzyme ([EI] Ͻ K D , [PEP] Ͻ K m ), ␣KG allosterically stimulates EI autophosphorylation. To our knowledge, this is one of the few examples of a small molecule metabolite being reported to both inhibit and stimulate the activity of the same enzyme depending on the experimental conditions (the other known case is ATP that can be a substrate or an allosteric inhibitor of phosphofructokinase) (17). The fact that the intracellular concentrations of EI, PEP, and ␣KG are modulated by the composition of the culturing medium (4, 14 -16) suggests that this interplay between allosteric stimulation and competitive inhibition of EI might be used by bacterial cells to regulate the phosphorylation state of PTS in response to a change in the extracellular environment.…”

Section: The Bacterial Phosphotransferase System (Pts) Is a Signal Trmentioning

confidence: 96%

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate

Nguyen¹,

Ghirlando

Venditti

2018

Journal of Biological Chemistry

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The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given EI's pivotal role in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on SOFAST NMR experiments, to investigate the effect of the smallmolecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that, at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when EI's oligomerization state is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes. _______________________________________ Enzyme I (EI) is the first protein of the bacterial phosphotransferase system (PTS), a signal transduction pathway that results in active sugar transport across the cell membrane (1-3). The PTS is initiated by phosphorylation of EI by the small molecule phosphoenolpyruvate (PEP). Phosphorylated EI transfers the phosphoryl group to the phosphocarrier protein HPr. Thereafter, the phosphoryl group is transferred to a sugar-specific enzyme II (EII), and finally to the incoming sugar ( Figure 1a). Recently, the small-molecule metabolite α-ketoglutarate (αKG) was shown to act as a competitive inhibitor of EI (inhibition constant, K I , ~ 2.2 mM) (4,5). The intracellular concentration of αKG varies considerably in response to a change in the availability of nitrogen source in the culturing medium (from 0.5 mM, in the presence of 10 mM NH 4 Cl, to 10 mM, in the absence of nitrogen source) (4). Thus, inhibition of EI by αKG has been proposed as a biochemical mechanism that links the uptake of sugars to the availability of nitrogen source (4,5 EI is a multidomain protein comprising a N-terminal domain (EIN, residues 1-249) that contains the phosphorylation site (His 189 ) and the binding site for HPr, and a C-terminal domain (EIC, residues 261-575) that is responsible for protein dimerization and contains the binding site for PEP and the competitive inhib...

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The Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate

Cited by 28 publications

References 37 publications

Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis inEscherichia coli

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate

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