Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.13.3 (histidine kinase)
 2,405 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Histidine kinase EnvZ, a transmembrane osmotic sensor for Escherichia coli, is a bifunctional enzyme having OmpR (its cognate response regulator) kinase and phosphorylated OmpR (OmpR-P) phosphatase activities. Its cytoplasmic domain consists of domain A responsible for dimerization of EnvZ, histidine phosphotransfer and phosphatase activities, and domain B responsible for ATP binding. Here, we have constructed a number of substitution mutations at the G2 box, one of the conserved motifs in domain B, and demonstrated that they influence the phosphatase activity of EnvZ over a wide range. The effects of ADP, a cofactor for the phosphatase activity, were found to be substantially different depending upon the mutations. The effects of these mutations were also examined in vivo using a chimeric Tar-EnvZ construct (Taz1-1), and the results agreed with the in vitro data for the phosphatase and kinase activities for all mutations. Using Taz1-1 carrying the T402A mutation, three independent intragenic suppressor mutations (T235M, S269L and E276K) were isolated, and all were found in domain A. Together, the present results demonstrate for the first time that domain A and domain B are functionally co-ordinated and topologically arranged in a specific manner. The G2 box may modulate the interaction between these two domains in response to extracellular osmolarity.
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PMID:The role of the G2 box, a conserved motif in the histidine kinase superfamily, in modulating the function of EnvZ. 1213 13

EnvZ, a histidine kinase, and its cognate response regulator OmpR of Escherichia coli are responsible for adaptation to external osmotic changes by regulating the levels of the outer membrane porin proteins, OmpF and OmpC. The osmosensor, EnvZ, has dual enzymatic functions with OmpR kinase and OmpR-P phosphatase. Here, we demonstrate that the cytoplasmic kinase domain of EnvZ (EnvZc) and OmpR are able to form a 1:1 complex detected by native PAGE. This indicates that two OmpR molecules can bind to one EnvZc dimer. As this 1:1 EnvZc/OmpR complex is formed even in the presence of a large excess of EnvZc, OmpR binding to EnvZc is co-operative. The complex formation is also observed between EnvZc and phosphorylated OmpR for the phosphatase reaction. OmpR-P bound to EnvZc was readily released upon the addition of OmpR, indicating that OmpR and OmpR-P can compete for the binding to EnvZ. On the basis of these results, a model is discussed to explain how cellular OmpR-P concentrations are regulated in response to medium osmolarity.
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PMID:Formation of the stoichiometric complex of EnvZ, a histidine kinase, with its response regulator, OmpR. 1245 14

EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein-labelled OmpR (Fl-OmpR) with EnvZc was determined to be 1.96 +/- 0.28 micro M. We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl-OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 +/- 0.25 micro M and 0.89 +/- 0.14 micro M, respectively, under the conditions used. The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl-OmpR or Fl-OmpR-P as well as with OmpR or OmpR-P as a substrate. We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.
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PMID:Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator. 1245 15

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.
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PMID:Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX. 1248 44

In Escherichia coli, PhoR is the histidine kinase of the phosphate regulon. It has been postulated that PhoR may function as a phospho-PhoB phosphatase. Experiments with four precise phoR deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain. This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.
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PMID:Genetic and biochemical studies of phosphatase activity of PhoR. 1253 89

We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator. A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene.
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PMID:Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus. 1256 8

The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg(2+) depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg(2+), in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states.
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PMID:Mutational analysis of the residue at position 48 in the Salmonella enterica Serovar Typhimurium PhoQ sensor kinase. 1261 57

EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.
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PMID:Cysteine-scanning analysis of the dimerization domain of EnvZ, an osmosensing histidine kinase. 1275 42

Two-component systems, which are comprised of a single histidine-aspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidine-aspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF(3)(-)-activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF(3)(-) activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators.
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PMID:The NMR solution structure of BeF(3)(-)-activated Spo0F reveals the conformational switch in a phosphorelay system. 1287 49

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.
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PMID:A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium. 1461 62


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