共查询到20条相似文献,搜索用时 31 毫秒
1.
Piszczek G Lee JC Tjandra N Lee CR Seok YJ Levine RL Peterkofsky A 《Archives of biochemistry and biophysics》2011,507(2):332-342
Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI(Ntr). Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4°C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI(Ntr) are evaluated. 相似文献
2.
《Journal of molecular biology》2019,431(12):2331-2342
There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTSsugar) and one for nitrogen regulation (PTSNtr), that utilize proteins enzyme Isugar (EIsugar) and HPr, and enzyme INtr (EINtr) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein–protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EINtr:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EINtr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EINtr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EINtr. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes. 相似文献
3.
Susan Jahn Bart R. Haverkorn van Rijsewijk Uwe Sauer Katja Bettenbrock 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):2879-2889
To investigate a possible role of the nitrogen-PTS (PTSNtr) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTSNtr, on different carbon sources. The PTSNtr is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EINtr (encoded by ptsP) and NPr (encoded by ptsO) to EIIANtr (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates. The ΔptsP and the ΔptsO strain showed the same behavior as the parent strain. As the phosphorylation level of EIIANtr in these mutants differed significantly from that of the parent strain, phosphorylation of EIIANtr obviously is not important for its function. During growth in minimal medium with defined carbon sources, EIIANtr was always completely phosphorylated in LJ110. Significant amounts of dephosphorylated EIIANtr were only visible in strains lacking EINtr or NPr. mRNA expression studies on glucose revealed a downregulation of genes encoding TCA cycle enzymes when EIIANtr was absent. 13C-flux analyses confirmed higher fluxes towards acetate and lower fluxes in the TCA cycle in the ptsN mutants but additionally hinted to a slightly but significantly increased flux through the pyruvate dehydrogenase complex (PDH). During growth on succinate the ΔptsN strain accumulated mutations in rpoS, while no rpoS mutants were observed for the ΔptsN-O strain. This hints to an additional function of NPr during growth with succinate. 相似文献
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6.
Yue Zhen Li Dan Wang Xue Ying Feng Jian Jiao Wen Xin Chen Chang Fu Tian 《Applied and environmental microbiology》2016,82(4):1305-1315
The nitrogen phosphotransferase system (PTSNtr) consists of EINtr, NPr, and EIIANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EINtr and NPr to EIIANtr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EINtr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIANtr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIANtr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EINtr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTSNtr in symbiosis was also observed when related PTSNtr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTSNtr in addition to the negative role of unphosphorylated EIIANtr. 相似文献
7.
Many Proteobacteria possess the paralogous PTSNtr, in addition to the sugar transport phosphotransferase system (PTS). In the PTSNtr phosphoryl‐groups are transferred from phosphoenolpyruvate to protein EIIANtr via the phosphotransferases EINtr and NPr. The PTSNtr has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTSNtr plays a role in potassium homeostasis. In particular, EIIANtr binds to and stimulates activity of a two‐component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high‐affinity K+ transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTSNtr. The pho regulon, which comprises more than 30 genes, is activated by the two‐component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIANtr are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild‐type level in mutants deficient for EIIANtr phosphorylation suggesting that non‐phosphorylated EIIANtr modulates pho. Protein interaction analyses reveal binding of EIIANtr to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIANtr is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli. 相似文献
8.
In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTSNtr). In this PTSNtr, the protein HPr is phosphorylated on histidine-22 by the enzyme EINtr and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EINtr, both proteins were purified and the activity of EINtr was measured. Experimentally determined kinetic parameters of EINtr activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EINtr. Binding experiments using the isolated GAF domain of EINtr (EIGAF) showed that it is the domain responsible for detection of glutamine. EINtr activity was not affected by α-ketoglutarate, and no binding between the EIGAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EINtr phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine). 相似文献
9.
The nitrogen‐related phosphoenolpyruvate phosphotransferase system (PTSNtr) is involved in controlling ammonia assimilation and nitrogen fixation. The additional role of PTSNtr as a regulatory link between nitrogen and carbon utilization in Escherichia coli is assumed to be closely related to molecular functions of IIANtr in potassium homeostasis. We have determined the crystal structure of IIANtr from Burkholderia pseudomallei (BpIIANtr), which is a causative agent of melioidosis. The crystal structure of dimeric BpIIANtr determined at 3.0 Å revealed that its active sites are mutually blocked. This dimeric state is stabilized by charge and weak hydrophobic interactions. Overall monomeric structure and the active site residues, Arg51 and His67, of BpIIANtr are well conserved with those of IIANtr enzymes from E. coli and Neisseria meningitides. Interestingly, His113 of BpIIANtr, which corresponds to a key residue in another phosphoryl group relay in the mannitol‐specific enzyme EIIA family (EIIAMtl), is located away from the active site due to the loop connecting β5 and α3. Combined with other differences in molecular surface properties, these structural signatures distinguish the IIANtr family from the EIIAMtl family. Since, there is no gene for NPr in the chromosome of B. pseudomallei, modeling and docking studies of the BpIIANtr–BpHPr complex has been performed to support the proposal on the NPr‐like activity of BpHPr. A potential dual role of BpHPr as a nonspecific phosphocarrier protein interacting with both sugar EIIAs and IIANtr in B. pseudomallei has been discussed. Proteins 2013. © 2013 Wiley Periodicals, Inc. 相似文献
10.
Kim HJ Lee CR Kim M Peterkofsky A Seok YJ 《Biochemical and biophysical research communications》2011,(3):556-561
Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIANtr of the nitrogen PTS regulates the intracellular concentration of K+ by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity. 相似文献
11.
Jingshan?Ren Sarah?Sainsbury Nick?S?Berrow David?Alderton Joanne?E?Nettleship David?K?Stammers Nigel?J?Saunders Raymond?J?Owens
Background
The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. 相似文献12.
Hurtado-Gómez E Abián O Muñoz FJ Hernáiz MJ Velázquez-Campoy A Neira JL 《Biophysical journal》2008,95(3):1336-1348
The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPrsc. By using fluorescence and circular dichroism, we first determined qualitatively that HPrsc and HPr9-30 did bind to EIsc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr9-30 and HPrsc for EIsc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr9-30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EIsc and HPrsc is enthalpy driven and in other species is entropy driven; further, the affinity of HPrsc for EIsc was smaller than in other species. However, the affinity of HPr9-30 for EIsc was only moderately lower than that of EIsc for HPrsc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS. 相似文献
13.
Zhen Song Xiaoyan Zheng Binsheng Yang 《Protein science : a publication of the Protein Society》2013,22(11):1519-1530
CopC is a periplasmic copper Chaperone protein that has a β‐barrel fold and two metal‐binding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site‐directed mutagenesis. The far‐UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The Δ<ΔG0element> obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the <ΔG0element> induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ/mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the <ΔG0element> induced by urea for Y79WW83L have a loss of 11.44 kJ/mol, but for Y79WW83F did a raise of 1.82 kJ/mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC. 相似文献
14.
Misha Golynskiy Sheng Li Virgil L. WoodsJr Seth M. Cohen 《Journal of biological inorganic chemistry》2007,12(5):699-709
The manganese transport regulator (MntR) of Bacillus subtilis is a metalloregulatory protein responsible for regulation of genes involved in manganese uptake by this organism. MntR belongs
to the iron-responsive DtxR family, but is allosterically regulated by manganese and cadmium ions. Having previously characterized
the metal binding affinities of this protein as well as the DNA-binding activation profiles for the relevant metal ions, we
have focused the current study on investigating the structural changes of MntR in solution upon binding divalent transition
metal ions. Deuterium exchange mass spectrometry was utilized to investigate the deuterium exchange dynamics between apo-MntR,
Co2+-MntR, Cd2+-MntR, and Mn2+-MntR. Comparing the rates of deuteration of each metal-bound form of MntR reveals that the N-terminal DNA-binding motif is
more mobile in solution than the C-terminal dimerization domain. Furthermore, significant protection from deuterium exchange
is observed in the helices that contribute metal-chelating amino acids to form the metal binding site of MntR. In contrast,
the bulk of the DNA-binding winged helix–turn–helix motif shows no difference in deuterium exchange upon metal binding. Mapping
of the deuteration patterns onto the crystal structures of MntR yields insight into how metal binding affects the protein
structure and complements earlier studies on the mechanism of MntR. Metal binding acts to rigidify MntR, thereby limiting
the mobility of the protein and reducing the entropic cost of DNA binding.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
Chang‐Ro Lee Young‐Ha Park Miri Kim Yeon‐Ran Kim Soyoung Park Alan Peterkofsky Yeong‐Jae Seok 《Molecular microbiology》2013,88(3):473-485
In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N‐terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so‐called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α‐ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability. 相似文献
16.
V. N. Uversky S. E. Permyakov I. I. Senin A. M. Cherskaya S. V. Shulga-Morskoy D. V. Zinchenko A. M. Alekseev A. A. Zargarov V. M. Lipkin P. P. Philippov E. A. Permyakov 《Russian Journal of Bioorganic Chemistry》2000,26(3):152-156
The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged
second and/or third Ca2+-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions
was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability
(its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar
structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca2+-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third
or simultaneously second and third Ca2+-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions.
For the communication I, see [1]. 相似文献
17.
Pseudomonas aeruginosa is an opportunistic human pathogen whose survival is aided by forming communities known as biofilms, in which cells are encased in a self‐produced matrix. We devised a mutant screen based on colony morphology to identify additional genes with previously unappreciated roles in biofilm formation. Our screen, which identified most known biofilm‐related genes, also uncovered PA14_16550 and PA14_69700, deletions of which abrogated and augmented biofilm formation respectively. We also identified ptsP, which encodes enzyme I of the nitrogen‐regulated phosphotransferase (PTSNtr) system, as being important for cyclic‐di‐GMP production and for biofilm formation. Further experiments showed that biofilm formation is hindered in the absence of phosphotransfer through the PTSNtr, but only in the presence of enzyme II (PtsN), the putative regulatory module of the PTSNtr. These results implicate unphosphorylated PtsN as a negative regulator of biofilm formation and establish one of the first known roles of the PTSNtr in P. aeruginosa. 相似文献
18.
Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics 下载免费PDF全文
Subrata Paul Hiroaki Ishida Leonard T. Nguyen Zhihong Liu Hans J. Vogel 《Protein science : a publication of the Protein Society》2017,26(5):946-959
Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo‐LipD is very similar to other four‐helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo‐LipD are more restricted than in apo‐LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo‐LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo‐LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS‐ACP from A. friuliensis that would allow the acyl‐CoA ligase to interact preferentially with the LipD instead of binding to the FAS‐ACP. 相似文献
19.
Aritra Chowdhury Aparajita Choudhury Victor Banerjee Rajat Banerjee K. P. Das 《Biopolymers》2014,101(5):549-560
α‐Crystallin is a multimeric eye lens protein having molecular chaperone‐like function which is crucial for lens transparency. The stability and unfolding‐refolding properties of α‐crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native‐like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α‐crystallin revealed dry native‐like core of α‐crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol?1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol?1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014. 相似文献
20.
Katharina Pflüger-Grau Max Chavarría Víctor de Lorenzo 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011