共查询到20条相似文献,搜索用时 15 毫秒
1.
Haikarainen T Thanassoulas A Stavros P Nounesis G Haataja S Papageorgiou AC 《Journal of molecular biology》2011,405(2):448-460
The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn2+, Mn2+, Ni2+, Co2+, Cu2+, and Mg2+) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg2+, were found to bind to the ferroxidase center similarly to Fe2+, with moderate affinity (binding constants between 0.1 × 105 M− 1 and 5 × 105 M− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers. 相似文献
2.
Donghyun Ka Dayoun Kim Gyeongyun Baek Euiyoung Bae 《Biochemical and biophysical research communications》2014
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes. 相似文献
3.
Surasak LimsuwanAnne Hesseling-Meinders Supayang Piyawan VoravuthikunchaiJan Maarten van Dijl Oliver Kayser 《Phytomedicine》2011,18(11):934-940
Rhodomyrtone from Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract has a strong antibacterial activity against the bacterial pathogen Streptococcus pyogenes. Our previous studies indicated that the bactericidal activity of rhodomyrtone might involve intracellular targets. In the present studies we followed a proteomics approach to investigate the mode of action of rhodomyrtone on S. pyogenes. For this purpose, S. pyogenes was cultivated in the presence of 0.39 μg/ml rhodomyrtone, which corresponds to 50% of the minimal inhibitory concentration. The results show that the amounts of various enzymes associated with important metabolic pathways were strongly affected, which is consistent with the growth-inhibiting effect of rhodomyrtone. Additionally, cells of S. pyogenes grown in the presence of rhodomyrtone produced reduced amounts of known virulence factors, such as the glyceraldehyde-3-phosphate dehydrogenase, the CAMP factor, and the streptococcal pyrogenic exotoxin C. Taken together, these findings indicate that rhodomyrtone has both antimicrobial and anti-infective activities, which make it an interesting candidate drug. 相似文献
4.
Doyoun Kim Sang Hyun Moh Hyejin Park Sangho Lee Kyeong Kyu Kim 《Biochemical and biophysical research communications》2010,391(1):431-11929
Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. 相似文献
5.
As part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H2O2 by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH· by growing the bacteria at acidic pH, or in the presence of an OH· scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host. 相似文献
6.
Ming Chen Rui Wang Liping Li Wanwen Liang Qiuhua Wang Ting Huang Chao Li Jian Li Xi Gan Aiying Lei Weiyi Huang Honglin Luo 《Cellular immunology》2014
The immunological effects of heat shock proteins (HSPs) had been found in humans and mice, but scarce data of endotoxin-free Hsp70 were reported in tilapia. In the current study, we reported that tHsp70 alone and antigen-tHsp70 compound increased the proliferations of lymphocytes and macrophages, significantly increased the NO release and phagocytotic ability of macrophages (p < 0.05), and enhanced the levels of immune-related genes in lymphocytes and macrophages in a dose- and/or time-dependent manner. On the other hand, tHsp70 not only helped to reduce the proliferation inhibitions induced by the ECP treatment, but also assisted antigens to enhance the vaccine-induced protection against Streptococcus iniae (p < 0.05). We described, for the first time, a critical role of endotoxin-free tHsp70 on activation of tilapia lymphocytes and macrophages post S. iniae exposure and its up-regulation effects on vaccine-induced protection. Our research highlights the immunological enhancement action of Hsp70 in teleost immunity. 相似文献
7.
Nina S. Ponomarenko Antony R. Marino Agnes Ostafin Edward J. Bylina James R. Norris Jr. 《生物化学与生物物理学报:生物膜》2009,1788(9):1822-1831
Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P43212, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C380, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades. 相似文献
8.
9.
Glauber P Arêas R?de BB Schuab Felipe PG Neves Rosana R Barros 《Memórias do Instituto Oswaldo Cruz》2014,109(7):935-939
Streptococcus pyogenes is responsible for a variety of infectious
diseases and immunological complications. In this study, 91 isolates of S.
pyogenes recovered from oropharynx secretions were submitted to
antimicrobial susceptibility testing, emm typing and pulsed-field
gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone,
levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin
was 15.4%, which is higher than previous reports from this area, while 20.9% of the
isolates were not susceptible to tetracycline. The macrolide resistance phenotypes
were cMLSB (10) and iMLSB (4). The ermB gene was predominant,
followed by the ermA gene. Thirty-two emm types and
subtypes were found, but five (emm1, emm4,
emm12, emm22, emm81) were detected in
48% of the isolates. Three new emm subtypes were identified
(emm1.74, emm58.14, emm76.7).
There was a strong association between emm type and PFGE clustering.
A variety of PFGE profiles as well as emm types were found among
tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial
resistant strains do not result from the expansion of one or a few clones. This study
provides epidemiological data that contribute to the development of suitable
strategies for the prevention and treatment of such infections in a poorly studied
area. 相似文献
10.
Célia V. Romão Edward P. Mitchell Sean McSweeney 《Journal of biological inorganic chemistry》2006,11(7):891-902
The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-Å resolution, and one soaked in an iron solution, to 1.6-Å resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage. 相似文献
11.
Yohta Fukuda Hiroyasu Koteishi Ryohei Yoneda Taro Tamada Hideto Takami Tsuyoshi Inoue Masaki Nojiri 《BBA》2014
The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition. 相似文献
12.
Imre Tör? Francesca Pacello Andrea Battistoni Kristina Djinovi?-Carugo 《Journal of molecular biology》2009,386(2):406-87
The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space. 相似文献
13.
We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome cz bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome cz. It was also shown that the rereduction rate of cytochrome cz by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center. 相似文献
14.
15.
Yu-Jin Kim Moon-Gi Jang Hae-Yong Noh Hye-Jin LeeJohan Sukweenadhi Jong-Hak KimSe-Yeong Kim Woo-Saeng KwonDeok-Chun Yang 《Gene》2014
Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H2O2) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses. 相似文献
16.
17.
In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes. 相似文献
18.
A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (PZn), the accessory Zn-BChl a (BZn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from PZn*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from BZn* to PZn, and the latter, the electron transfer from PZn to H. The angle between the transition dipoles of BZn and PZn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies PZn. 相似文献
19.
Tharangani Rathnayaka Minako TawaShihori Sohya Masafumi YohdaYutaka Kuroda 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(9):1902-1907
Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 °C. Soluble-GLuc remained fully folded until 40 °C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 °C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein. 相似文献
20.
Andrea C. RuthesYanna D. Rattmann Elaine R. CarboneroPhilip A.J. Gorin Marcello Iacomini 《Carbohydrate polymers》2012,87(2):1620-1627
Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had Mw 43.8 × 104 g mol−1 and RFP-Lr Mw 1.4 × 104 g mol−1. RFP-Lr had a (1 → 6)-linked α-d-Galp main-chain partially substituted at O-2 by nonreducing end-units of α-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially substituted at O-2 by nonreducing end-units of α-l-Fucp (2.8%) and β-d-Galp (14.5%), and partially methylated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate, myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and decreasing in TNF-α and IL-1β serum levels. 相似文献