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E B Waygood

Publications and source records attributed to E B Waygood.

At least 37 records · Page 2Linked to original sources
PubMed1993

The involvement of the arginine 17 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 His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Amino Acid Sequence↗
PubMed1993

Phosphoryl transfer between phosphorylated histidine-containing protein and histidine-containing protein is not autocatalytic.

Histidine-containing protein, HPr, is a phosphocarrier protein that is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated by enzyme I, and P-HPr transfers the phosphoryl group to the IIA domain of a number of sugar-specific enzyme II complexes. Autocatalytic phosphoryl transfer between P-HPr and HPr has recently been reported [van Dijk, A. A., Eisermann, R., Hengstenberg, W., & Robillard, G. T. (1991) Biochemistry 30, 2876-2882]. Our results show that this phosphoryl transfer is due to an unidentified contaminant of HPr preparations. The phosphoryl transfer activity is not present in all HPr preparations. When present, the phosphoryl transfer activity can be removed by further purification or destroyed over time by resuspension of HPr preparations in water. There is no autocatalytic phosphoryl transfer between P-HPr and HPr.

Bacterial Proteins↗
PubMed1993

Evaluation of mutagenesis for epitope mapping. Structure of an antibody-protein antigen complex.

The location and description of epitopes on proteins describe the basis of immunological specificity. The 2.8-A structure of the phosphocarrier protein, HPr from Escherichia coli, complexed to the Fab fragment of the monoclonal antibody, Jel42, has been determined. This allows the first comparison of epitope predictions from extensive site-directed mutagenesis experiments, coupled with biological activity studies (Sharma, S., Georges, F., Klevit, R. E., Delbaere, L. T. J., Lee, J. S., and Waygood, E. B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4877-4881), with those from x-ray analysis. There are 14 amino acid residues of E. coli HPr that interact with the Jel42 antigen-binding site. Nine of these were correctly assigned by the mutagenesis studies. Of the 5 remaining residues, Met-1 could not be altered; two others appear to have critical roles in determining protein conformation; the other 2 residues have a minimal effect on antibody binding since they are located on the periphery of the epitope with one face of their side chains in van der Waals contact with the antibody and the other face in contact with solvent. Four residues were incorrectly assigned to the epitope. These residues were located adjacent to epitope residues that were likely perturbed by these mutations. This study demonstrates that mutations which caused greater than 10-fold changes in antibody binding affinity were correctly assigned to the epitope by the mutagenesis experiments. Guidelines are also presented in order to minimize incorrect assignments.

Amino Acid Sequence↗
PubMed1991

Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy.

Analysis of the histidine-containing protein (HPr) from Escherichia coli by two-dimensional homonuclear and heteronuclear nuclear magnetic resonance techniques has been performed, extending the work originally reported [Klevit, R. E., Drobny, G. D., & Waygood, E. B. (1986) Biochemistry 25, 7760-7769; Klevit, R. E., & Drobny, G. P. (1986) Biochemistry 25, 7770-7773; Klevit, R. E., & Waygood, E. B. (1986) Biochemistry 25, 7774-7781]. Two-dimensional homonuclear total coherence spectroscopy (TOCSY) allowed for more complete assignments of the side-chain spin systems than had been possible in the original studies. As well, two-dimensional 15N-1H heteronuclear spectroscopy was used to resolve a number of ambiguities present in the homonuclear spectra due to resonance redundancies. These analyses led to the correction of a number of resonance assignments that were made with the spectra that could be collected with the technology that existed 6 years ago. In addition, amide exchange rates and 3JNH coupling constants have been measured, extending the original analysis and yielding new structural information. All these data have been used to reexamine the folding topology of E. coli HPr. Structure calculations showed that the topology derived from the earlier NMR data, i.e., a four-stranded beta-sheet with three alpha-helices running along one side of the sheet, was essentially unchanged, although at the present level of analysis, a well-defined "helix B" could not be established with high confidence. In addition, the data reported here revealed the existence of two slowly-exchanging side-chain hydroxyl protons belonging to Ser31 and Thr59. Their behavior strongly suggests that these side chains are involved in hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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↗
PubMed1991

Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

Amino Acid Sequence↗
PubMed1991

Sequences of the variable regions of three monoclonal antibodies specific for histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The variable regions of three monoclonal antibodies, Jel 42, Jel 44, and Jel 324, specific for the histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system have been sequenced from their respective mRNAs. The Vh gene families were deduced from the percent homology to the concensus gene sequences and the J gene and D gene usage was also analysed.

Amino Acid Sequence↗
PubMed1990

Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli.

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate-polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62,000; nagA, 45,000; nagB, 29,000. In addition, another product of Mr 44,000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weight (MWs) of the gene products derived from the sequence are as follows: nagA, 40,954; nagB, 29,657; nagC, 44,664; nagE, 68,356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27,181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: nagD nagC nagA nagB nagE.

Acetylglucosamine↗
PubMed1989

Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.

Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown. This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J. S., and Delbaere, L. T. J. (1988) J. Biol. Chem. 263, 2571-2574). Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding. The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4. There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%.

Antibodies, Monoclonal↗
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↗
PubMed1988

Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes.

Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins. These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system. The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues. Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.

Amino Acid Sequence↗
PubMed1988

Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus faecalis.

The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1-phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.

Amino Acid Sequence↗
PubMed1987

Tertiary structure of histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

The tertiary structure of the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been determined by x-ray diffraction at 2.8-A resolution. Initially, a partial structure was fitted to the multiple isomorphous replacement map and then least-squares refined by the Konnert/Hendrickson restrained parameter method (Konnert, J. H., and Hendrickson, W. A. (1980) Acta Crystallogr. A36, 344-350) and finally, a subsequent map was computed by use of the phase combination method of Read (Read, R. J. (1986) Acta Crystallogr. A42, 140-149). More of the protein structure was located in the latter map. The procedure of model building, least-squares refinement, and electron density map recalculation was repeated until the tertiary structure of HPr was obtained. The overall structure of HPr consists of four beta-strands, three helical regions, and four beta-turns. At the active center, the His15 imidazole interacts with one oxygen atom of the alpha-carboxyl C terminus of the polypeptide chain; the conserved Arg17 side chain interacts with the other oxygen atom of the alpha-carboxyl C terminus as well as with the side chain of Glu85. This is the first x-ray analysis of a protein of the phosphoenolpyruvate:sugar phosphotransferase system. Furthermore, this work represents a protein structure which has been solved by starting with a model that represented only one-third of the scattering matter.

Escherichia coli↗
PubMed1987

HPr/HPr-P phosphoryl exchange reaction catalyzed by the mannitol specific enzyme II of the bacterial phosphotransferase system.

The mannitol specific Enzyme II of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli catalyzes an exchange reaction in which a phosphoryl moiety is transferred from one molecule of the heat stable phosphocarrier protein HPr to another. An assay was developed for measuring this reaction. Unlabeled phospho-HPr and 125I-labeled free HPr were incubated together in the presence of Enzyme IImtl, and production of 125I-labeled phospho-HPr was measured. The reaction was concentration-dependent with respect to Enzyme IImtl and did not occur in its absence. The reaction occurred in the absence of Mg2+ in the presence of 10 mM EDTA. Treatment of Enzyme IImtl with the histidyl reagent diethylpyrocarbonate inactivated it with respect to the exchange reaction. Levels of N-ethylmaleimide which inactivate Enzyme IImtl with respect to both P-enolpyruvate-dependent phosphorylation of mannitol and mannitol/mannitol-1-P transphosphorylation did not affect its activity in the exchange reaction; however, treatment with another sulfhydryl reagent, p-chloromercuribenzoate, resulted in partial inactivation. The pH optimum for the Enzyme IImtl-catalyzed exchange reaction was about 7.5. Enzyme I and the glucose specific Enzyme III, two other E. coli phosphotransferase system proteins which, like Enzyme IImtl, interact directly with HPr, were also shown to catalyze 125I-HPr/HPr-P phosphoryl exchange.

Bacterial Proteins↗
PubMed1987

Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium.

Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a beta-sheet structure.

Antibodies, Monoclonal↗
PubMed1986

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments.

Two-dimensional NMR studies at 500 MHz have been performed on the histidine-containing protein (HPr) from Escherichia coli. HPr is one of the phosphocarrier proteins involved in the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is responsible for the concomitant phosphorylation and translocation of a number of sugars. Sequential resonance assignments of HPr are complete. The conventional method of sequential assignments involving J-correlated spectroscopy (COSY) and nuclear Overhauser spectroscopy (NOESY) has been supplemented by optimized relayed coherence transfer spectroscopy (RELAY) to help overcome the spectral overlap that is inevitable in the spectra of proteins the size of HPr. RELAY experiments were performed in H2O to obtain NH-C beta H connectivities and in D2O to obtain C alpha H-C gamma H connectivities. The abundance of relayed coherence transfer peaks in the two experiments greatly aided in the assignment process of the complicated protein spectrum. The assignments lay the groundwork for the determination of the solution structure of HPr, as described in the accompanying paper [Klevit, R. E., & Waygood, E. B. (1986) Biochemistry (third paper of three in this issue)].

Amino Acid Sequence↗
PubMed1986

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR.

Sequence-specific resonance assignments of the 1H NMR spectrum of the 85-residue histidine-containing phosphocarrier protein (HPr) are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. Additional side-chain assignments have been made with long-range coherence transfer experiments [Klevit, R. E., & Drobny, G. P. (1986) Biochemistry (second paper of three in this issue)]. In this paper, the NMR assignments were used to determine the secondary structure and the tertiary folding of HPr in solution. The secondary structural elements of the protein were determined by visual inspection of the pattern of nearest-neighbor nuclear Overhauser effects (NOEs) and the presence of persistent amide resonances. Escherichia coli HPr consists of four beta-strands, three alpha-helices, four reverse turns, and several regions of extended backbone structure. Long-range NOEs, especially among side-chain protons, were used to determine the tertiary structure of the protein by use of the secondary structural components. The four beta-strands form a single antiparallel beta-pleated sheet. The hydrophobic faces of the alpha-helices interact to form a hydrophobic core and sit above the hydrophobic face of the beta-sheet, forming an open-face beta-sheet sandwich structure. The active site histidine, His-15, is on a short kinked segment of backbone that is accessible to the solvent. The positively charged phosphorylation site (His-15 and Arg-17) interacts with the negatively charged carboxyl terminus of the protein (Glu-85).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗