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PubMed1995

Purification and characterization of a small membrane-associated sugar phosphate phosphatase that is allosterically activated by HPr(Ser(P)) of the phosphotransferase system in Lactococcus lactis.

In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate. The enzyme exhibits a Ka for S46D HPr of 15 microM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.

Allosteric Regulation↗

PubMed1995

Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli. Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant.

Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

Amino Acid Sequence↗

PubMed2002

A new family of phosphotransferases with a P-loop motif.

In most Gram-positive bacteria, catabolite repression is mediated by a bifunctional enzyme, the histidine-containing protein kinase/phosphatase (HprK/P). Based either on its primary sequence or on its recently solved three-dimensional structure, no straightforward homology with other known proteins was found. However, we showed here that HprK/P exhibits a restricted homology with an unrelated phosphotransferase, the phosphoenolpyruvate carboxykinase. This includes notably two consecutive Asp residues from the phosphoenolpyruvate carboxykinase active site, whose equivalent residues were mutated in Bacillus subtilis HprK/P. Characterization of the corresponding mutants emphasizes the crucial role of these Asp residues in the HprK/P functioning. Furthermore, superimposition of HprK/P and phosphoenolpyruvate carboxykinase active sites supports the view that both enzymes bear significant resemblance in their overall mechanism of functioning showing that these two enzymes constitute a new family of phosphotransferases.

Amino Acid Motifs↗

PubMed2002

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines, His-15 of HPr and His-65 of IIA(Mtl), are in close spatial proximity, and a pentacoordinate phosphoryl transition state can be readily accommodated with no change in protein-protein orientation and only minimal perturbations of the backbone immediately adjacent to the histidines. Comparison with two previously solved structures of complexes of HPr with partner proteins of the phosphotransferase system, the N-terminal domain of enzyme I (EIN) and enzyme IIA(Glucose) (IIA(Glc)), reveals a number of common features despite the fact that EIN, IIA(Glc), and IIA(Mtl) bear no structural resemblance to one another. Thus, entirely different underlying structural elements can form binding surfaces for HPr that are similar in terms of both shape and residue composition. These structural comparisons illustrate the roles of surface and residue complementarity, redundancy, incremental build-up of specificity and conformational side chain plasticity in the formation of transient specific protein-protein complexes in signal transduction pathways.

Amino Acid Sequence↗

PubMed2003

The molecular basis of the expansive substrate specificity of the antibiotic resistance enzyme aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2". The role of ASP-99 as an active site base important for acetyl transfer.

The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6'-aminoglycoside phosphotransferase-2" (AAC(6')- aminoglycoside phosphotransferase-2", capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6')-Ie, is the only member of the large AAC(6') subclass known to modify fortimicin A and catalyze O-acetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6')-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6')-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.

Acetyltransferases↗

PubMed2005

Transient state kinetics of enzyme IICBGlc, a glucose transporter of the phosphoenolpyruvate phosphotransferase system of Escherichia coli: equilibrium and second order rate constants for the glucose binding and phosphotransfer reactions.

During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA(Glc) and Enzyme IICB(Glc), the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA(Glc) to IICB(Glc) to Glc) and also the second order rate constants for the transfer from phospho-IIA(Glc) to molecularly cloned IIB(Glc), the soluble, cytoplasmic domain of IICB(Glc). The rate constants for the forward and reverse phosphotransfer reactions between IIA(Glc) and IICB(Glc) were 3.9 x 10(6) and 0.31 x 10(6) m(-1) s(-1), respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB(Glc) and Glc was 3.2 x 10(6) m(-1) s(-1). From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIA(Glc) to the phosphorylation site Cys of IIB(Glc) or IICB(Glc) were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB(Glc) were invariably found to contain endogenous, firmly bound Glc (estimated K'(D) approximately 10(-7) m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB(Glc) has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.

Enzyme Stability↗

PubMed2005

Solution NMR structure of the 48-kDa IIAMannose-HPr complex of the Escherichia coli mannose phosphotransferase system.

The solution structure of the 48-kDa IIA(Man)-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system has been solved by NMR using conjoined rigid body/torsion angle-simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIA(Man) is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIA(Man). The interaction surface on IIA(Man) is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 A(2). The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic, and charged residues. The active site histidines, His-10 of IIA(Man) and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-epsilon2 atom of His-10 and the N-delta1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIA(glucose), and IIA(mannitol), reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins.

Bacterial Proteins↗

PubMed2006

Structure of the full-length enzyme I of the phosphoenolpyruvate-dependent sugar phosphotransferase system.

Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5A reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35A between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.

Binding Sites↗

PubMed1990

Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins.

The bacterial phosphotransferase system (PTS) is the major transport system for many carbohydrates that are phosphorylated concomitantly with the translocation step through the membrane (group translocation). It consists of two general proteins, enzyme I and histidine protein (HPr), and a series of more than 15 substrate-specific enzymes II (EII). The sequences of several of these derived from Gram-positive and Gram-negative bacteria were compared, which allowed the possible identification of the following functional domains: membrane-bound pore, substrate-binding site, linker domains, transphosphorylation domain and primary phosphorylation site. Several EIIs have been analysed in the meantime, also by topological tests, by sequential deletion of the corresponding structural genes, and by construction of intergenic hybrids between different domains of several EIIs. These data suggest evolutionary relationships between different EIIs; they also enable a general model to be constructed of EIIs as carbohydrate transport systems, phosphotransferases, chemoreceptors in chemotaxis and as part of a global regulatory network.

Amino Acid Sequence↗

PubMed2004

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase.

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

Amino Acid Sequence↗

PubMed1991

Mutations in the aphA-2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside-phosphotransferases strongly reduce aminoglycoside resistance.

Aminoglycoside-phosphotransferases contain several conserved amino acid sequence motifs. Using hydroxylamine we have obtained five independent missense mutations within the aphA-2 gene of transposon Tn5. Four of the mutations dramatically reduced antibiotic resistance. Two were identical and included the replacement of His-188 with Tyr. One other resulted from the replacement of Gly-189 with Asp. These three mutations map within the first of the conserved motifs. The replacement of Asp-261 with Asn maps to the third of these structural motifs. A mutation diminishing but not eliminating aminoglycoside resistance resulted from replacement of the conserved Val-36 with Met. By site-directed mutagenesis three additional mutants were obtained: His-188 was replaced with Leu and Ser, and Arg-211 within the second conserved motif was substituted by Gly. All three showed reduced levels of resistance to kanamycin. Our results show that these conserved motifs are essential for the biological activity of aminoglycoside phosphotransferases.

Amino Acid Sequence↗

PubMed1976

Phosphorylation of intracellular fructose in Bacillus subtilis mediated by phosphoenolpyruvate-1-fructose phosphotransferase.

Intracellular fructose provided by the sorbitol pathway in Bacillus subtilis can be phosphorylated by the phosphenolpyruvate-1-fructose phosphotransferase which is known to mediate a vectorial metabolism. The fate of this intracellular fructose was studied using mutants lacking either the fructose 1-phosphate pathway or the fructose 6-phosphate pathway. It was shown that the phosphoenolpyruvate-dependent phosphorylation needs a prior exit of the sugar into the medium, this exit being probably catalysed by a transport system. A low affinitiy intracellular phosphenolpyruvate phosphotransferase system was found, which seems to be devoid of a physiological role.

Bacillus subtilis↗

PubMed1978

Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes.

Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.

DNA-Directed RNA Polymerases↗

PubMed1980

Mitochondrial ATP:AMP phosphotransferase from beef heart: purification and properties.

ATP:AMP phosphotransferase has been purified 186-fold from beef heart mitochondria in an overall yield of 30%. The purified enzyme, Mr 31 500, has a specific activity of 800 U/mg and was found to be a homogeneous protein by constancy of specific activity across the elution peak following passage through an Affi-Gel Blue agarose column and by dodecylsulfate gel electrophoresis. Isoelectrofocusing shows a minor peak of activity that arises again when the major peak is re-focused. The pI of the enzyme is 9.30 and the optimum pH of activity is 5.8 in the forward reaction (formation of ADP) and 8.0 in the reverse reaction. The enzyme requires the presence of a divalent metal ion, namely Mg2+, Co2+, Ca2+ Mn2+, Ni2+ and Zn2+ in decreasing order of activity and is influenced by NaCl. It shows rigorous specificity for AMP (dAMP) was phosphate acceptor but is rather unspecific for the triphosphate donor since, UTP, CTP, ITP and GTP may substitute for ATP (dATP). ATP and MgATP (dATP was not tested) bind to the enzyme with dissociation constants of 0.122 and 0.132 mM respectively, while AMP (dAMP was not tested), UTP, CTP, ITP and GTP binding could not be detected by the dialysis equilibrium technique used. Amino acid analysis gave the following composition as residues: 26 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 24 proline, 19 glycine, 25 alanine, 17 valine, 8 methionine, 14 isoleucine, 31 leucine, 5 tyrosine, 9 phenylalanine, 6 histidine, 25 lysine, 15 arginine, 4 half cystine (no free sulfhydryls) and no tryptophan, giving a total of 286 residues. The purified mitochondrial adenylate kinase is not a contaminating heart cytosolic enzyme. ATP:AMP phosphotransferase was extracted from beef heart cytosol depleted of mitochondria and brought to homogeneity. Determinations of pI, Mr and amino acid composition showed that the heart mitochondrial and cytosolic adenylate kinases are different isozymes.

Adenylate Kinase↗

PubMed1983

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities.

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.

Acetyltransferases↗

PubMed1984

The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

Adenylate Kinase↗

PubMed1984

Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system.

Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.

Binding Sites↗

PubMed1985

Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides. The coupling between transport and phosphorylation in inside-out vesicles.

The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the literature. We have tested the coupling by analysing the kinetics of fructose phosphorylation by inside-out vesicles of Rhodopseudomonas sphaeroides. We conclude that fructose indeed has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The Km of the phosphorylation reaction is 8 microM. The diffusion of fructose into the vesicle is a reaction that is also catalysed by the components of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a Km of 4 mM and is rate-limiting over a large external fructose concentration range. In summary there is no phosphorylation without transport, but there is transport without phosphorylation.

Biological Transport, Active↗