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PubMed1993

Mapping of the binding interfaces of the proteins of the bacterial phosphotransferase system, HPr and IIAglc.

Enzyme IIAglc and HPr are central regulatory and phosphocarrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of bacteria. During phosphoryl transfer from phosphoenolpyruvate to glucose, phosphate is transferred from HPr to enzyme IIAglc. In order to characterize the binding interfaces of the two proteins during phosphate transfer, 15N-edited and 15N-filtered NMR experiments have been recorded for the complex of enzyme IIAglc and HPr from Bacillus subtilis. Uniformly 15N-labeled enzyme IIAglc and nonlabeled HPr were used in these studies. Residues which undergo significant chemical shift changes upon complex formation have been identified for both proteins. The binding interfaces of the two proteins, suggested by the observed chemical shift changes, involve predominantly hydrophobic surfaces near the active site His-15 of HPr and the phosphoryl acceptor His-83 of IIAglc.

Bacillus subtilis↗

PubMed1991

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.

Bacterial Proteins↗

PubMed1994

Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity.

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.

Aminoglycosides↗

PubMed1991

Exchange of phosphoryl groups between HPr molecules of the phosphoenolpyruvate-dependent phosphotransferase system is an autocatalytic process.

HPr, a central component of the phosphoenolpyruvate-dependent phosphotransferase system, can exist in Escherichia coli in a phosphorylated (PHPr) and a nonphosphorylated form. We show that, beside the normal transfer of the phosphoryl group from PHPr to enzymes II and III, PHPr can phosphorylate other HPr molecules in an autocatalytic exchange reaction. The reaction is very fast but is inhibited by labeling the protein with Bolton-Hunter reagent. We demonstrate that the exchange reaction can be used to determine the delta G degree of the phosphoryl group of mutant forms of PHPr relative to wild-type PHPr. Two HPr mutants were constructed by site-directed mutagenesis, HPr P11E and HPr E68A. Both show altered phosphoryl group potentials but show no significantly altered KM or Vmax values compared to wild-type HPr, illustrating the sensitivity of the exchange process. The exchange reaction does not occur between HPr from E. coli and HPr from Staphylococcus carnosus.

Autoradiography↗

PubMed1991

Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased. The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG. The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence. Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII. When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme. The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII [Lolkema J. S., ten Hoeve-Duurkens, R. H., Swaving Dijkstra, D., & Robillard, G. T. (1991) Biochemistry (preceding paper in this issue)]. Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant. No effect on the binding properties was observed. In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr.

Cytoplasm↗

PubMed1994

Active-site labeling of an aminoglycoside antibiotic phosphotransferase (APH(3')-IIIa).

The aminoglycoside antibiotics are inactivated by modifying enzymes that are now widely distributed in many pathogenic bacteria. This situation threatens the continued use of these clinically important drugs. We have undertaken studies to understand the molecular mechanism of aminoglycoside resistance, and we report the affinity labeling of the enterococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-dependent irreversible inactivation of enzyme activity with a binding constant, Ki, of 0.406 mM and a rate of maximal inactivation, kmax, of 0.086 min-1. Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reaction of APH(3')-IIIa with [14C]FSBA showed that inactivated APH(3')-IIIa incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-inactivated APH(3')-IIIa resulted in the identification of two peptide peaks with highly increased absorbance at 260 nm, indicative of covalent labeling with FSBA. Analysis by electrospray ionization mass spectrometry and Edman degradation revealed two tryptic peptides, Val31-Lys44 and Leu34-Arg49, which incorporated the FSBA label at Lys33 and Lys44, respectively. This establishes the importance of the N-terminal region of APHs in ATP binding, a region of these enzymes which has heretofore not been considered for involvement in substrate binding.

Adenosine↗

PubMed1982

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: mechanism of phosphoryl-group transfer from phosphoenolpyruvate to HPr.

The mechanism of phosphoryl-group transfer from phosphoenolpyruvate (PEP) to HPr, catalyzed by enzyme I of the Escherichia coli PEP-dependent phosphotransferase system, has been studied in vitro. Steady-state kinetics and isotope exchange measurements revealed that this reaction cannot be described by a classical ping-pong mechanism although phosphoenzyme I acts as an intermediate. The kinetic data indicate that HPr and PHPr occupy binding sites on enzyme I that do not overlap with the binding sites for PEP and pyruvate. As a result, binding interactions between HPr and enzyme I exist regardless of their phosphorylated state. A general mechanism is presented that describes the phosphorylation of HPr. The physiological implications of this mechanism are discussed.

Escherichia coli↗

PubMed1983

Bacterial phosphoenolpyruvate-dependent phosphotransferase system. Mechanism of the transmembrane sugar translocation and phosphorylation.

The phosphoryl-group transfer from PHPr to glucose or alpha-methylglucose and from glucose 6-phosphate to these same sugars catalyzed by membrane-bound EIIBGlc of the bacterial phosphoenolpyruvate-dependent phosphotransferase system has been studied in vitro. Kinetic measurements revealed that both the phosphorylation reaction and the exchange reaction proceed according to a ping-pong mechanism in which a phosphorylated membrane-bound enzyme II acts as an obligatory intermediate. The occurrence of a phospho-IIBGlc/IIIGlc has been physically demonstrated by the production of a glucose 6-phosphate burst from membranes phosphorylated by phosphoenolpyruvate, HPr, and EI. The observation of similar second-order rate constants for the production of sugar phosphate starting with different phosphoryl-group donors confirms the catalytic relevance of the phosphoenzyme IIBGlc intermediate. The in vitro results, together with data published by other investigators, have led to a model describing sugar phosphorylation and transport in vivo.

Bacteria↗

PubMed1985

Characterization of phosphorylated histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system, when phosphorylated, contains a 1-phosphohistidinyl (1-P-histidinyl) residue (His-15). The properties of this 1-P-histidinyl residue were investigated by using phospho-HPr (P-HPr), P-HPr-1, and P-HPr-2. HPr-1 and HPr-2 are deamidated forms of HPr produced by boiling. In addition, HPr-1 produced during frozen storage was investigated. Both pH and temperature dependencies of the rate of hydrolysis of the phosphoryl group of the 1-P-histidinyl residue were investigated. The results show that the 1-P-histidinyl residue in HPr and HPr-1 has significantly different properties from free 1-P-histidine and that these differences are attributable to the active-site residues Glu-66 and Arg-17 and the pK of the imidazole group of the 1-P-histidinyl residue in P-HPr. The 1-P-histidinyl residue in P-HPr and P-HPr-1 shows a greater lability at physiological pH than the free amino acid. A proposal for the active site of P-HPr is made on the basis of these results and the recently obtained tertiary structure. In contrast, the hydrolysis properties of the 1-P-histidinyl residue in P-HPr-2 were similar to those obtained for either free 1-P-histidine or denatured P-HPr. The loss of activity that is associated with boiling HPr was shown to be due to HPr-2 formation as HPr-1 was found to be fully active.

Bacterial Proteins↗

PubMed1986

Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium.

The glucose-specific membrane permease (IIGlc) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates active transport and concomitant phosphorylation of glucose. The purified permease has been phosphorylated in vitro and has been isolated (P-IIGlc). A phosphate to protein stoichiometry of between 0.6 and 0.8 has been measured. Phosphoryl transfer from P-IIGlc to glucose has been demonstrated. This process is, however, slow and accompanied by hydrolysis of the phosphoprotein unless IIIGlc, the cytoplasmic phosphoryl carrier protein specific to the glucose permease (IIGlc) of the PTS, is added. Addition of unphosphorylated IIIGlc resulted in rapid formation of glucose 6-phosphate with almost no hydrolysis of P-IIGlc accompanying the process. A complex of IIGlc and IIIGlc could be precipitated from bacterial cell lysates with monoclonal anti-IIGlc immunoglobulin. The molar ratio of IIGlc:IIIGlc in the immunoprecipitate was approximately 1:2. Analytical equilibrium centrifugation as well as chemical cross-linking showed that purified IIGlc itself is a dimer (106 kDa), consisting of two identical subunits. These results suggest that the functional glucose-specific permease complex comprises a membrane-spanning homodimer of IIGlc to which four molecules of IIIGlc are bound on the cytoplasmic face.

Biological Transport, Active↗

PubMed1986

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system has two sites of phosphorylation per dimer.

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli has been reported to contain one phosphorylation site per dimer and thus operates by either a half of the sites or a flip-flop mechanism [Misset, O., & Robillard, G. T. (1982) Biochemistry 21, 3136-3142; Hoving, T., ten Hoeve-Duurkens, R., & Robillard, G. T. (1984) Biochemistry 23, 4335-4340]. In this paper, the determination of two phosphorylation sites per dimer of enzyme I was made by using a number of different methods. In some experiments, less than two sites per dimer were found, but a concomitant loss in enzyme I activity was also found. The phosphorylated residue in enzyme I was shown to have the properties expected for a N3-phosphohistidinyl residue.

Buffers↗

PubMed1986

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr.

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1987

Bacterial phosphoenolpyruvate-dependent phosphotransferase system: association state of membrane-bound mannitol-specific enzyme II demonstrated by inactivation.

The quaternary structure of the membrane-bound mannitol permease (EIIMtl) of the bacterial phosphotransferase system in Escherichia coli has been investigated in the membrane by using the radiation inactivation method. The experiments reveal two distinct but interconvertible forms of the permease. The first state is a dimer, and the second state consists of a less active higher molecular weight complex involving the dimer. The equilibrium between these two forms in the membrane can be shifted by changing the pH. At pH 8.1 the dimer is the dominant form. Decreasing the pH results in increased binding of a regulatory protein to the dimer, thus increasing the amount of the higher molecular weight form involving the dimer. Cross-linking EIIMtl in situ, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, resulted in the formation of two cross-linked forms. One is the dimer, and the other has a higher molecular weight. Two-dimensional electrophoresis using a reversible cross-linker revealed no other protein except EIIMtl in these complexes.

Alcohol Dehydrogenase↗

PubMed1988

Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier.

The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.

Chromatography, High Pressure Liquid↗

PubMed1988

Sequence of cloned enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:N-acetylglucosamine phosphotransferase system of Escherichia coli.

In Escherichia coli, N-acetylglucosamine (nag) metabolism is joined to glycolysis via three specific enzymes that are the products of the nag operon. The three genes of the operon, nagA, nagB, and nagE, were found to be carried by a colicin plasmid, pLC5-21, from a genomic library of E. coli [Clarke, L., & Carbon, J. (1976) Cell (Cambridge, Mass.) 9,91-99]. The nagE gene that codes for enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was sequenced. The nagE sequence is preceded by a catabolite gene activator protein binding site and ends in a putative rho-independent termination site. The amino acid sequence determined from this DNA sequence shows 44% homology to enzymes IIglucose and IIIglucose of the PTS. Enzyme IIN-acetylglucosamine, which has 648 amino acids and a molecular weight of 68,356, contains a histidine at residue 569 which is homologous to the active site of IIIglc. Sequence homologies with enzymes IIglucose, II beta-glucoside, and IIsucrose indicate that residues His-190, His-213, and His-295 of enzyme IInag are also conserved and that His-190 is probably the second active site histidine. Other sequence homologies among these enzymes II suggest that they contain several sequence transpositions. Preliminary models of the enzymes II are proposed.

Amino Acid Sequence↗

PubMed1990

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:mannitol phosphotransferase system.

We have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EIIMtl, the phospho group from [(R)-16O,17O,18O]PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EIIMtl must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EIIMtl intermediates have been demonstrated biochemically, our results imply that EIIMtl is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EIIMtl intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein [Pas, H. H., & Robillard, G. T. (1988) Biochemistry 27, 5835-5839].

Binding Sites↗

PubMed1990

Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent.

The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted [Saier, M.H. (1980) J. Supramol. Struct. 14, 281-294]. The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed. Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions. The dimer is favored over the monomer at high ionic strength and basic pH. Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization. A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer. Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions. The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3. The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed. The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction.

Bacterial Proteins↗

PubMed1990

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit.

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.

Binding Sites↗