Crystal Structure of Enzyme I of the Phosphoenolpyruvate Sugar Phosphotransferase System in the Dephosphorylated State

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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Reconstitution Studies Using the Helical and Carboxy-Terminal Domains of Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System

Biochemistry ◽

10.1021/bi991680p ◽

1999 ◽

Vol 38 (47) ◽

pp. 15470-15479 ◽

Cited By ~ 20

Author(s):

Peng-Peng Zhu ◽

Roman H. Szczepanowski ◽

Neil J. Nosworthy ◽

Ann Ginsburg ◽

Alan Peterkofsky

Keyword(s):

Phosphotransferase System ◽

Carboxy Terminal ◽

Enzyme I ◽

Terminal Domains

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Structural insight into glucose repression of the mannitol operon

Scientific Reports ◽

10.1038/s41598-019-50249-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Mangyu Choe ◽

Huitae Min ◽

Young-Ha Park ◽

Yeon-Ran Kim ◽

Jae-Sung Woo ◽

...

Keyword(s):

Glucose Repression ◽

Phosphorylation Site ◽

Phosphotransferase System ◽

E Coli ◽

Carbon Compounds ◽

Structural Insight ◽

Enzyme I ◽

Insight Into ◽

Repressor Activity

Abstract Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within the Enterobacteriaceae family.

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A novel phosphoprotein dependent on the bacterial phosphoenolpyruvate–sugar phosphotransferase system

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-022 ◽

1983 ◽

Vol 61 (2-3) ◽

pp. 150-153 ◽

Cited By ~ 6

Author(s):

E. Bruce Waygood ◽

Roshan L. Mattoo

Keyword(s):

Escherichia Coli ◽

Salmonella Typhimurium ◽

Growth Conditions ◽

Phosphotransferase System ◽

Crude Extracts ◽

Enzyme I ◽

Novel Protein

A protein has been found by isoelectricfocusing and autoradiography in Escherichia coli and Salmonella typhimurium which was phosphorylated by enzyme I and an histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate–sugar phosphotransferase system (PTS). This protein was not factor IIIglc nor was it specifically induced by fructose. Its presence in soluble crude extracts was dependent upon growth conditions; however, the two bacteria had different patterns and amounts in respect to this novel protein. The protein was present in S. typhimurium SB2950 which has an extensive deletion through the pts operon, thus indicating that it must be coded for elsewhere on the genome.

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PTS-Mediated Regulation of the Transcription Activator MtlR from Different Species: Surprising Differences despite Strong Sequence Conservation

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000369619 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 94-105 ◽

Cited By ~ 8

Author(s):

Philippe Joyet ◽

Meriem Derkaoui ◽

Houda Bouraoui ◽

Josef Deutscher

Keyword(s):

Bacillus Subtilis ◽

Lactobacillus Casei ◽

Sequence Conservation ◽

Geobacillus Stearothermophilus ◽

Transcription Activator ◽

Phosphotransferase System ◽

Transcription Regulator ◽

Regulatory Domains ◽

Genes Encoding ◽

Enzyme I

The hexitol <smlcap>D</smlcap>-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the <smlcap>D</smlcap>-mannitol-specific PTS components and <smlcap>D</smlcap>-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIBGat- and an EIIAMtl-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTSMtl components, which inactivate MtlR by phosphorylating its EIIBGat- or EIIAMtl-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIBMtl-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIAMtl-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIBMtl domain.

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