Biomedical subjects

A Peterkofsky

Publications and source records attributed to A Peterkofsky.

At least 19 recordsLinked to original sources

PubMed1998

Autophosphorylation of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system requires dimerization.

Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition. In vitro autophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.

Dimerization↗

PubMed1998

Topography of the interaction of HPr(Ser) kinase with HPr.

The phosphocarrier protein, HPr, from Gram-positive organisms and mycoplasmas is a substrate for an ATP-dependent kinase that phosphorylates serine 46. In Gram-negative organisms, the corresponding HPr is not phosphorylated on serine 46 and the ATP-dependent kinase is absent. To determine the specificity requirements for phosphorylation of Mycoplasma capricolum HPr, a chimera in which residues 43-57 were replaced by the Escherichia coli sequence was constructed. The chimeric protein folded properly, but was not phosphorylated on either serine 46 or histidine 15. A dissection of the region required for phosphorylation specificity was carried out by further mutagenesis. The deficiency in phosphorylation at histidine 15 was localized primarily to the region including residues 51-57. Activity studies revealed that residues 48, 49, and 51-53 are important for recognition of M. capricolum HPr by its cognate HPr(Ser) kinase. The characteristics of this region suggest that the kinase-HPr interaction occurs mainly through a hydrophobic region. Molecular modeling comparisons of M. capricolum HPr and the chimeric construct provided a basis for interpreting the results of the activity assays.

Amino Acid Sequence↗

PubMed1998

A promiscuous binding surface: crystal structure of the IIA domain of the glucose-specific permease from Mycoplasma capricolum.

BACKGROUND: The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a bacterial and mycoplasma system responsible for the uptake of some sugars, concomitant with their phosphorylation. The sugar-specific component of the system, enzyme II (EII),consists of three domains, EIIA, EIIB and EIIC. EIIA and ELLB are cytoplasmic and EIIC is an integral membrane protein that contains the sugar-binding site. Phosphoenolpyruvate (PEP) provides the source of the phosphoryl group, which is transferred via several phosphoprotein intermediates, eventually being transferred to the internalized sugar. Along the pathway, EIIA accepts a phosphoryl group from the phosphocarrier protein HPr and transfers it to EIIB. The structure of the glucose-specific EIIA (EIIAglc) from Mycoplasma capricolum reported here facilitates understanding of the nature of the interactions between this protein and its partners. RESULTS: The crystal structure of EIIAglc from M. capricolum has been determined at 2.5 A resolution. two neighboring EIIAglc molecules associate with one another in a front-to-back fashion, such that Glu149 of one molecule forms electrostatic interactions with the active-site histidine residues, His90 and His75, of the other. Glu149 is therefore considered to mimic the interaction that a phosphorylated histidine of a partner protein makes with EIIA. Another interaction, an ion pair between the active-site Asp94 and Lys168 of a neighboring molecule, may be analogous to the interaction between Asp94 of EIIAglc and Arg17 of HPr. Analysis of molecular packing in this crystal, and in the crystals of two other homologous proteins from Escherichia coli and Bacillus subtilis, reveals that in all cases active-site hydrophobic residues are involved in crystal contacts, but in each case a different region of the neighboring molecule is involved. The transition-state complexes of M. capricolum EIIAglc with HPr and EIIBglc have been modeled; in each case, different structural units are shown to interact with EIIAglc. Many of the interactions are hydrophobic with no sequence specificity. The only specific interaction, other than that formed by the phosphoryl group, involves ion pairs between two invariant aspartate residues of EIIAglc and arginine/lysine residues of HPr or EIIBglc. CONCLUSIONS: The non-discriminating nature of the hydrophobic interactions that EIIAglc forms with a variety of partners may be a consequence of the requirement for interaction with a variety of proteins that show no sequence or structural similarity. Nevertheless, specificity is provided by an ion-pair interaction that is enhanced by the apolar nature of the interface.

Amino Acid Sequence↗

PubMed1998

Phosphorylation destabilizes the amino-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

Thermal stabilities of enzyme I (63 562 M(r) subunit, in the Escherichia coli phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), and a cloned amino-terminal domain of enzyme I (EIN; 28 346 Mr) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD) at pH 7.5. EIN expressed in a delta pts E. coli strain showed a single, reversible, two-state transition with Tm = 57 degrees C and an unfolding enthalpy of approximately 140 kcal/mol. In contrast, monomeric EIN expressed in a wild-type strain (pts+) had two endotherms with Tm congruent with 50 and 57 degrees C and overall delta H = 140 kcal/mol and was converted completely to the more stable form after five DSC scans from 10 to 75 degrees C (without changes in CD: approximately 58% alpha-helices). Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis. Dephospho-enzyme I (monomer right arrow over left arrow dimer) exhibited endotherms for C- and N-terminal domain unfolding with Tm = 41 and 54 degrees C, respectively. Thermal unfolding of the C-terminal domain occurred over a broad temperature range ( approximately 30-50 degrees C), was scan rate- and concentration-dependent, coincident with a light scattering decrease and Trp residue exposure, and independent of phosphorylation. Reversible thermal unfolding of the nonphosphorylated N-terminal domain was more cooperative, occurring from 50 to 60 degrees C. DSC of partially phosphorylated enzyme I indicated that the amino-terminal domain was destabilized by phosphorylation (from Tm = 54 to approximately 48 degrees C). A decrease in conformational stability of the amino-terminal domain of enzyme I produced by phosphorylation of the active-site His 189 has the physiological consequence of promoting phosphotransfer to the phosphocarrier protein, HP(r).

Calorimetry, Differential Scanning↗

PubMed1998

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.

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy. The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa 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 Ndelta1-H tautomer, and its Nepsilon2 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 chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state.

Bacterial Proteins↗

PubMed1997

Topology of allosteric regulation of lactose permease.

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Allosteric Regulation↗

PubMed1997

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Allosteric Regulation↗

PubMed1997

Expression, purification, and characterization of enzyme IIA(glc) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum.

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the overexpressed gene was characterized as an active phosphoacceptor from HPr with a higher pI than previously described EIIAs. M. capricolum EIIA was unreactive with antibodies directed against the corresponding proteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-sheet structural content: temperature-induced changes in ellipticity at 205 nm showed that the protein undergoes reversible, two-state thermal unfolding with Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I (64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)], respectively, in sedimentation equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate highly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsic tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these genes are located diametrically opposite in the M. capricolum chromosome.

Chromosome Mapping↗

PubMed1997

Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system.

The interaction between the approximately 30 kDa N-terminal domain of enzyme I (EIN) and the approximately 9.5 kDa histidine-containing phosphocarrier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting us to follow the chemical shift changes of the backbone NH and 15N resonances of EIN upon complex formation by recording a series of 1H-15N correlation spectra of uniformly 15N-labeled EIN in the presence of increasing amounts of HPr at natural isotopic abundance. The equilibrium association constant derived from analysis of the titration data is approximately 1.5 x 10(5) M(-1), and the lower limit for the dissociation rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garrett, D. S., Seok, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identified the binding surface of EIN in contact with HPr. This surface is primarily located in the alpha domain and involves helices H1, H2, and H4, as well as the hinge region connecting helices H2 and H2'. The data also indicate that the active site His 15 of HPr must approach the active site His 189 of EIN along the shallow depression at the interface of the alpha and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range 1H-15N correlation spectrum) of His 189, which is located at the N-terminus of helix H6 in he alpha/beta domain, are only minimally perturbed upon complexation, indicating that His 189 (in the absence of phosphorylation) does not undergo any significant conformational change or change in pK(a) value upon HPr binding. On the basis of results of this study, as well as a previous study which delineated the interaction surface for EI on HPr [van Nuland, N. A. J., Boelens, R., Scheek, R. M., & Robillard, G. T. (1995) J. Mol. Biol. 246, 180-193], a model for the EIN/HPr complex is proposed in which helix 1 (residues 16-27) and the helical loop (residues 49-53) of HPr slip between the two pairs of helices constituting the alpha domain of EIN. In addition, we suggest a functional role for the kink between helices H2 and H2' of EIN, providing a flexible joint for this interaction to take place.

Bacterial Proteins↗

PubMed1997

Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR.

The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy. Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II. To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths. Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments. The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms. The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I. A comparison with the recently solved X-ray structure of EIN [Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1996) Structure 4, 861-872] indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3. His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.

Amino Acid Sequence↗

PubMed1996

The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr.

BACKGROUND: The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) transports exogenous hexose sugars through the membrane and tightly couples transport with phosphoryl transfer from PEP to the sugar via several phosphoprotein intermediates. The phosphate group is first transferred to enzyme I, second to the histidine-containing phosphocarrier protein HPr, and then to one of a number of sugar-specific enzymes II. The structures of several HPrs and enzymes IIA are known. Here we report the structure of the N-terminal half of enzyme I from Escherichia coli (EIN). RESULTS: The crystal structure of EIN (MW approximately 30 kDa) has been determined and refined at 2.5 A resolution. It has two distinct structural subdomains; one contains four alpha helices arranged as two hairpins in a claw-like conformation. The other consists of a beta sandwich containing a three-stranded antiparallel beta sheet and a four-stranded parallel beta sheet, together with three short alpha helices. Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein. CONCLUSIONS: The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.

Amino Acid Sequence↗

PubMed1996

Importance of the region around glycine-338 for the activity of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change glycine-338 to a variety of other amino acid residues. Fermentation tests indicated that glycine-338 could be mutated to alanine with no gross loss in phosphotransferase activity, while mutation to valine, glutamic acid, aspartic acid, arginine, histidine, or asparagine led to significant loss of activity. An expression vector for enzyme I was mutated to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. Analysis of phosphorylation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation, and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences the autophosphorylation rather than the phosphoryl transfer activity of enzyme I.

Amino Acid Sequence↗

PubMed1996

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Amino Acid Sequence↗

PubMed1996

Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum.

The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr. Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M.genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.

Acetate Kinase↗

PubMed1995

Structural evidence for the evolutionary divergence of mycoplasma from gram-positive bacteria: the histidine-containing phosphocarrier protein.

BACKGROUND: The three-dimensional structures of histidine-containing phosphocarrier protein (HPr), a member of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), have been determined from Gram-negative and Gram-positive bacteria. The structure of HPr reported here for Mycoplasma capricolum is the first protein structure to be determined for this class of organism. Comparative structural studies with the bacterial proteins highlight sequence-structure correlations relevant to proposals about the evolutionary origin of mycoplasmas. RESULTS: The crystal structure of HPr from M. capricolum has been determined and refined at 1.8 A resolution, revealing the same overall fold as that of other HPrs of known structure. However, mycoplasma HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria. As in HPrs from Bacillus subtilis and Escherichia coli, the phosphoryl group carrier (His15) forms the N-terminal cap of a helix, but in contrast to the other crystal structures, the side chain of the adjacent Arg17 is conformationally disordered. A sulfate ion interacts with Ser46, a residue known to be phosphorylated in a regulatory manner. CONCLUSIONS: The greater degree of structural similarity of the M. capricolum HPr to HPrs from Gram-positive rather than Gram-negative bacteria is consistent with the proposal that mycoplasma evolved from Gram-positive bacteria. The proposal that no major conformational transition is required for phosphorylation of the active-site histidine is reinforced by comparing the crystal structures with and without an anion in the active site. The conformational disorder of the Arg17 side chain suggests that its guanidinium group does not have to form specific interactions with other protein groups before phosphorylation at His15. The association of a sulfate ion with Ser46 serves as a model for HPr(Ser46-P). As there is no evidence of a conformational change accompanying Ser46 phosphorylation, the inhibitory effect of this event may be attributable to altered surface electrostatics.

Amino Acid Sequence↗

PubMed1995

The Escherichia coli adenylyl cyclase complex: requirement of PTS proteins for stimulation by nucleotides.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

Adenylyl Cyclases↗

PubMed1995

A dual mechanism for regulating cAMP levels in Escherichia coli.

In Escherichia coli, inorganic orthophosphate regulates cAMP levels by acting at two separate loci. First, adenylyl cyclase activity measured in permeabilized cells of E. coli is substantially stimulated by physiological concentrations of inorganic phosphate. This stimulation does not require the presence of cAMP phosphodiesterase activity. Second, measurements of cAMP phosphodiesterase activity in permeabilized cells show a dose-dependent inhibition of that activity by inorganic orthophosphate. A model is proposed in which inorganic orthophosphate serves as a multifaceted regulator of cAMP levels by both stimulating synthesis and inhibiting degradation of the nucleotide.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Organization, sequence and regulation of expression of the murine Hoxa-7 gene.

The genomic sequence of Hoxa-7 (encoding the HOXa-7 homeobox protein), including the coding region (0.7 kb), flanked by a 5'-upstream region (2.8 kb), a 3'-downstream region (1 kb) and interrupted by an intron (995 bp), was determined. Northern blot analysis indicated the transcript size of Hoxa-7 to be 2.1-2.4 kb. Reverse transcription-PCR and primer extension analysis established the 5'-boundary of the mRNA to be in the region 1166 nt upstream from the start codon. Transient transfection of various Hoxa-7::cat constructs in NIH 3T3 cells was used to characterize the transcriptional activity of the 5'-flanking region of the gene. Constructs containing 544, 274 and 71 bp of the region upstream from the transcription start point (tsp) exhibited 78, 203 and 407%, respectively, of the activity shown by a control construct containing 739 bp of the upstream region. These data suggested the presence of negative regulatory elements in the region from 544 to 71 bp upstream from the tsp.

3T3 Cells↗