HPr kinase/phosphorylase, the sensor enzyme of catabolite repression in Gram-positive bacteria: structural aspects of the enzyme and the complex with its protein substrate.
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ABSTRACT:
SUBMITTER: Nessler S
PROVIDER: S-EPMC164879 | biostudies-literature | 2003 Jul
REPOSITORIES: biostudies-literature
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HPr kinase/phosphorylase, the sensor enzyme of catabolite repression in Gram-positive bacteria: structural aspects of the enzyme and the complex with its protein substrate.
Nessler Sylvie S Fieulaine Sonia S Poncet Sandrine S Galinier Anne A Deutscher Josef J Janin Joël J
Journal of bacteriology 20030701 14
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Project description:We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.
| S-EPMC111324 | biostudies-literature
Project description:Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of beta-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of beta-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.
| S-EPMC22949 | biostudies-literature
Project description:Carbon catabolite repression (CCR) is a regulatory phenomenon implemented by bacteria to hierarchically organize carbohydrate utilization in order to achieve maximal growth. CCR is likely of great importance to Streptococcus pneumoniae because the human host sites inhabited by this pathogen represent complex carbohydrate environments. In this species, inactivation of the prototypical Gram-positive CCR master regulator, ccpA, attenuates virulence in mice but does not relieve CCR of most metabolic enzymes, suggesting CcpA-independent CCR mechanisms predominate. Here we show the activities of three transcriptional regulators constitute the majority of transcriptional CCR of galactose metabolism operons. We determined seryl-phosphorylated histidine phosphocarrier protein (HPr-Ser?P)-mediated regulation is a major CCR mechanism and an essential activity in the pneumococcus, as an HPr point mutation abolishing HPrK/P-dependent phosphorylation was not tolerated nor was deletion of hprk/p. The HPr-Ser?P phosphomimetic mutant HPr S46D had reduced phosphotransferase system transport rates and limited induction of CCR-repressed genes. These results support a model of pneumococcal CCR in which HPr-Ser?P directly affects the activity of CcpA while indirectly affecting the activity of pathway-specific transactional regulators. This report describes the first CcpA-independent CCR mechanism identified in the pneumococcus and the first example of lethality from loss of HPr-Ser?P-mediated CCR in any species.
| S-EPMC4836947 | biostudies-literature
Project description:Carbohydrate catabolite repression (CCR) in Streptococcus mutans can be independent of catabolite control protein A (CcpA) and requires specific components of phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) permeases. Here, the effects of various ptsH (HPr) and hprK (HPr kinase/phosphatase) mutations on growth and CCR were evaluated. An hprKV265F mutation, which enhanced Ser46 phosphorylation of HPr, inhibited growth on multiple PTS sugars. A ptsHS46A mutation reversed the effects of hprKV265F in most cases. A strain carrying a ptsHS46D mutation, which mimics HPr(Ser-P), presented with more severe growth defects than the hprKV265F mutant. The hprKV265F mutant enhanced CCR of the fruA and levD operons, a phenotype reversible by the ptsHS46A mutation. The effects of the hprKV265F mutation on fruA and levD expression were independent of CcpA, but dependent on ManL (IIAB(Man)) and, to a lesser extent, on FruI (IIABC(Fru)), in a carbohydrate-specific fashion. Expression of the Bacillus subtilis ptsG gene in the manL mutant did not restore CCR of the lev or fru operons. The hprKV265F mutation inhibited growth on cellobiose and lactose, but only the transcription of the cel operon was decreased. Thus, in S. mutans, serine-phosphorylated HPr functions in concert with particular PTS permeases to prioritize carbohydrate utilization by modulating sugar transport and transcription of catabolic operons.
| S-EPMC2927710 | biostudies-literature
Project description:Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas , have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar-nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the (4)C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients.
| S-EPMC3985747 | biostudies-literature
Project description:UnlabelledBackgroundThe thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.ResultsWe report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.ConclusionCo-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.
| S-EPMC3526391 | biostudies-literature
Project description:Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.
| S-EPMC6311466 | biostudies-literature
Project description:HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and sigma(35) gene transcripts 4 h ahead of the parent strain, but there was no effect on sigma(28) synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.
| S-EPMC135351 | biostudies-literature
Project description:In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.
| S-EPMC4366281 | biostudies-literature
Project description:A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 +/- 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.
| S-EPMC516805 | biostudies-literature