Overproduction and rapid purification of the Phosphoenolpyruvate: Sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli

Reddy, P.G.; Fredd-Kuldell, Natalie; Liberman, E S; Peterkofsky, Alan

doi:10.1016/1046-5928(91)90069-u

Cited by 37 publications

(35 citation statements)

References 17 publications

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“…Protein was expressed and purified as described (47). Crystals of the WT enzyme were obtained at room temperature by vapor diffusion in hanging drops.…”

Section: Methodsmentioning

confidence: 99%

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

Teplyakov

Lim

Zhu

et al. 2006

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

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179

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sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

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“…Protein was expressed and purified as described (47). Crystals of the WT enzyme were obtained at room temperature by vapor diffusion in hanging drops.…”

Section: Methodsmentioning

confidence: 99%

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

Teplyakov

Lim

Zhu

et al. 2006

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

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179

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“…The fractions containing substantial amounts of FrsA were pooled and concentrated in a 3 K Macrosep centrifugal concentrator (Pall Gelman Laboratory, Ann Arbor, MI). The concentrated pool was further purified on a hydroxy- , and EIIB Glc were purified as described previously (12,15,16).…”

Section: Methodsmentioning

confidence: 99%

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Koo

Yoon

Lee

et al. 2004

Journal of Biological Chemistry

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The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA Glc (IIA Glc )) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA Glc -binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA Glc , with the highest affinity of any protein thus far shown to interact with IIA Glc . Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the ␥-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA Glc regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA. The bacterial phosphoenolpyruvate(PEP)1 :sugar phosphotransferase system (PTS) plays an important role in the transport of a variety of sugar substrates. This system catalyzes phosphorylation coupled to translocation of numerous simple sugars across the cytoplasmic membrane. The PTS is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein, HPr, which are used for all sugars, and, in addition, some sugar-specific components collectively known as enzymes II (1). The glucose-specific enzyme II of Escherichia coli consists of two components: soluble enzyme IIA Glc (IIA Glc ) and membrane-bound enzyme IICB Glc (IICB Glc ). Thus, glucose transport in E. coli involves three soluble PTS components (EI, HPr, and IIA Glc , encoded by the ptsHIcrr operon) and one membrane-bound protein, enzyme IICB Glc (encoded by the ptsG gene). Glucose uptake entails sequential phosphoryl transfer via the PTS, as follows: phosphoenolpyruvate (PEP) 3 EI 3 HPr 3 IIA Glc 3 IICB Glc 3 glucose. Components of the PTS also participate in several regulatory mechanisms (1). Catabolite repression allows for the preferential utilization of sugars transported by the PTS. Consequently, when E. coli are cultured in a medium containing both glucose and a non-PTS sugar, the glucose is consumed first. The currently accepted mechanism for this effect is that, when PTS sugars are transported, the steady-state condition of IIA Glc is mainly in the dephospho-form. The unphosphorylated form of IIA Glc inhibits transport of non-PTS sugars such as lactose, maltose, melibiose, and raffinose by interacting with transporters for these sugars (a process termed inducer exclusion) (1, 2-4). Other allosteric regulatory functions of IIA Glc incl...

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“…Materials and methods: Phosphorylation of EIN: Enzyme I, "N EIN, and HPr were prepared as described by Reddy et al (1991) and Garrett et al (1997aGarrett et al ( , 1997b. respectively.…”

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confidence: 99%

Tautomeric state and pK_a of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system

et al. 1998

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Abstract:The phosphorylated form of the N-terminal domain of enzyme I of the phosphoeno1pyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range IH-I5N correlation spectroscopy. The active site His 189 is phosphorylated at the Ne2 position and has a pK, of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the N61-H tautomer, and its Ne2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a x2 angle in the g + conformer in the unphosphorylated state to the g-conformer in the phosphorylated state. Keywords

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Overproduction and rapid purification of the Phosphoenolpyruvate: Sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli

Cited by 37 publications

References 17 publications

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Tautomeric state and pK_a of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system

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Overproduction and rapid purification of the Phosphoenolpyruvate: Sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli

Cited by 37 publications

References 17 publications

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

Tautomeric state and pKa of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system

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Tautomeric state and pK_a of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system