Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.     

Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIA^Glc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIA^Glc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIA^Glc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIA^Glc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIA^Glc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]–[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIA^Glc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.     

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

SUMMARY

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.     

Unraveling the evolutionary history of the phosphoryl-transfer chain of the phosphoenolpyruvate:phosphotransferase system through phylogenetic analyses and genome context

Background  

The phosphoenolpyruvate phosphotransferase system (PTS) plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP) and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS.     

Regulation of E. coli glycogen phosphorylase activity by HPr

Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.     

Histidine phosphorylation in biological systems

Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl-CoA synthetase. In the PTS, the NMR structure of the phosphohistidine moiety of the phosphohistidine-containing protein was determined but no X-ray structures of phosphohistidine forms of PTS proteins have been elucidated. There have been crystal structures of a few phosphohistidine-containing proteins determined: nucleoside diphosphate kinase, succinyl-CoA synthetase, a cofactor-dependent phosphoglycerate mutase and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum. A common theme for these stable phosphohistidines is the occurrence of ion-pair hydrogen bonds (salt bridges) involving the non-phosphorylated nitrogen atom of the histidine imidazole ring with an acidic amino acid side chain.     

Genetic Engineering of the Phosphocarrier Protein NPr of the Escherichia coli Phosphotransferase System Selectively Improves Sugar Uptake Activity

The Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) in prokaryotes mediates the uptake and phosphorylation of its numerous substrates through a phosphoryl transfer chain where a phosphoryl transfer protein, HPr, transfers its phosphoryl group to any of several sugar-specific Enzyme IIA proteins in preparation for sugar transport. A phosphoryl transfer protein of the PTS, NPr, homologous to HPr, functions to regulate nitrogen metabolism and shows virtually no enzymatic cross-reactivity with HPr. Here we describe the genetic engineering of a "chimeric" HPr/NPr protein, termed CPr14 because 14 amino acid residues of the interface were replaced. CPr14 shows decreased activity with most PTS permeases relative to HPr, but increases activity with the broad specificity mannose permease. The results lead to the proposal that HPr is not optimal for most PTS permeases but instead represents a compromise with suboptimal activity for most PTS permeases. The evolutionary implications are discussed.     

The phosphotransferase system of Streptomyces coelicolor.

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.     

Mutational Analysis of the Role of HPr in Listeria monocytogenes

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers

Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.     

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.     

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.     

Genetic dissection of specificity determinants in the interaction of HPr with enzymes II of the bacterial phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli

The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (EIIs). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and EI of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the EIIs of E. coli. In contrast, activation of the E. coli BglG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 16, 17, 20, 24, 27, 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive EIIs.     

Comparative genomic analyses of the bacterial phosphotransferase system.

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer.     

细菌磷酸转移酶系统(PTS)的组成与功能研究进展

细菌磷酸烯醇丙酮酸(phosphoenolpyruvate,PEP)-磷酸转移酶系统(phosphotransferase system,PTS)广泛存在于细菌、真菌和一些古细菌中,但不存在于动植物中。PTS由酶I (EI)、组氨酸磷酸载体蛋白(HPr或NPr)和酶II复合物等磷酸转移酶组成,既具有催化转运功能,又具有非常广泛的调节功能。PTS主要是通过磷酸级联反应将各种糖及其衍生物进行磷酸化然后运输到胞内。其不仅参与碳、氮中心代谢,调节铁、钾稳态,调控某些病原体的毒力,还能介导应激反应。在这些不同的调节过程中,信号由PTS组分的磷酸化状态提供,而该磷酸化状态根据PTS底物的可用性和细胞代谢状态的变化而变化。本文对细菌中磷酸转移酶系统的组成和调控网络进行综述,以期为PTS的整体调控机制及其对细菌整体代谢影响的研究提供参考依据。