The phosphoenolpyruvate-dependent glucose–phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell

Gabor, Elisabeth; Göhler, Anna-Katharina; Kosfeld, Anne; Staab, Ariane; Kremling, Andreas; Jahreis, Knut

doi:10.1016/j.ejcb.2011.04.002

Cited by 55 publications

(53 citation statements)

References 60 publications

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“…These PTS systems transfer a phosphate from phosphoenolpyruvate to a carbohydrate during import (28). PTS systems have been shown to be important in regulating carbohydrate flux in bacterial cells (29) including enteroinvasive E. coli and S. enterica serovar Typhimurium. In this particular system, the HADSF member likely dephosphorylates the imported xylitol, mannitol, or similar phosphosugar for metabolic utilization, although high activity with other alcohol sugars may point to substrate variability in this PTS system.…”

Section: Resultsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

139

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Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

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Section: Resultsmentioning

confidence: 99%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

132

139

View full text Add to dashboard Cite

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“…The small peptide SgrT inhibits the transport function of the PTS transporter probably by binding to the linker region connecting the EIIC Glc and EIIB Glc domains. Indeed, replacement of a proline (Pro-384) present in a conserved region (-L-K-T-P-G-R-E-D-) of the linker in E. coli PtsG with an arginine prevented the repressive effect of SgrT on glucose utilization (154). Surprisingly, the conserved linker motif is also found in the PTS permeases PtsG, GamP, and NagP of B. subtilis, although there is no evidence for the presence of an SgrT homologue in firmicutes.…”

Section: Fig 5 Pts-catalyzed Glucose Uptake and The Eiiamentioning

confidence: 99%

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Deutscher

Aké

Derkaoui

et al. 2014

Microbiol Mol Biol Rev

342

374

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SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

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“…Many recent studies have revealed that the regulation of ptsG expression is very complex and takes place at both the transcriptional and the posttranscriptional levels (reviewed in references 3 and 12). In addition, there also exists a direct regulation of transport activity that prevents intracellular hexose phosphate stress (10,32). In the absence of glucose, ptsG expression is repressed by Mlc.…”

mentioning

confidence: 99%

Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12

Göhler¹,

Staab²,

Gabor³

et al. 2011

Journal of Bacteriology

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The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICB Glc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICB Glc , Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICB Glc , by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. D espite the fact that Escherichia coli K-12 has been an important model organism for many decades, the function of Ͼ20% of all putative open reading frames in its genome remains unknown. Moreover, in many cases, no obvious sequence similarities with previously characterized proteins exist. The mtfA gene (formerly named yeeI), a monocistronic gene located at 44.1 min in the E. coli chromosome, has clearly belonged to this group. Intensive sequence similarity searches revealed that orthologs of MtfA (mnemonic for Mlc titration factor A) exist in more than 100 proteobacteria of the alpha, beta, and gamma subdivisions. In a previous report (2), we were able to demonstrate that E. coli MtfA is a cytoplasmic protein that binds to the carboxy-terminal part of the Mlc repressor protein (mnemonic for "making large colonies" [gene, dgsA]) with a very high affinity. Mlc is one of the global regulators of carbohydrate metabolism in E. coli and is especially involved in the regulation of the ptsG gene expression, which encodes the glucose transporter EIICB Glc . The EIICB Glc together with the cytoplasmic protein EIIA Glc , encoded by the crr gene (as part of the ptsHI-crr operon), forms the glucosespecific phosphoenolpyruvate (PEP)-dependent carbohydratephosphotransferase system (glucose-PTS). Both proteins take part in a phosphorylation cascade, which begins with an autophosphorylation reaction of the so-called enzyme I (EI; gene, ptsI) at the expense...

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The phosphoenolpyruvate-dependent glucose–phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell

Cited by 55 publications

References 60 publications

Panoramic view of a superfamily of phosphatases through substrate profiling

Panoramic view of a superfamily of phosphatases through substrate profiling

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12

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