耐辐射奇球菌dps突变株的构建和蛋白功能初步研究

Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H₂O₂)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H₂O₂(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。     

The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure,iron binding,and hydroxyl-radical scavenging properties

Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H2O2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H2O2, the major component of the plant defense system when challenged by the bacterium.     

Helicobacter hepaticus Dps protein plays an important role in protecting DNA from oxidative damage

The ferritin-like DNA-binding protein from starved cells (Dps) family proteins are present in a number of pathogenic bacteria. Dps in the enterohepatic pathogen, Helicobacter hepaticus is characterized and a H. hepaticus dps mutant was generated by insertional mutagenesis. While the wild type H. hepaticus cells were able to survive in an atmosphere containing up to 6.0% O₂, the dps mutant failed to grow in 3.0% O₂, and it was also more sensitive to oxidative reagents like H₂O₂, cumene hydroperoxide and t-butyl hydroperoxide. Upon air exposure, the dps^-  cells had more damaged DNA than the wild type; they became coccoid or lysed and they contained ∼6-fold higher amount of 8-oxoguanine (8-oxoG) DNA lesions than wild type cells. Purified H. hepaticus Dps was shown to be able to bind both iron and DNA. The iron-loaded form of Dps protein had much greater DNA binding ability than the native Dps or the iron-free Dps.     

The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.     

The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site

The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.     

The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties

Listeria innocua Dps (DNA binding protein from starved cells) affords protection to DNA against oxidative damage and can accumulate about 500 iron atoms within its central cavity through a process facilitated by a ferroxidase center. The chemistry of iron binding and oxidation in Listeria Dps (LiDps, formerly described as a ferritin) using H(2)O(2) as oxidant was studied to further define the mechanism of iron deposition inside the protein and the role of LiDps in protecting DNA from oxidative damage. The relatively strong binding of 12 Fe(2+) to the apoprotein (K(D) approximately 0.023 microM) was demonstrated by isothermal titration calorimetry, fluorescence quenching, and pH stat experiments. Hydrogen peroxide was found to be a more efficient oxidant for the protein-bound Fe(2+) than O(2). Iron(II) oxidation by H(2)O(2) occurs with a stoichiometry of 2 Fe(2+)/H(2)O(2) in both the protein-based ferroxidation and subsequent mineralization reactions, indicating complete reduction of H(2)O(2) to H(2)O. Electron paramagnetic resonance (EPR) spin-trapping experiments demonstrated that LiDps attenuates the production of hydroxyl radical by Fenton chemistry. DNA cleavage assays showed that the protein, while not binding to DNA itself, protects it against the deleterious combination of Fe(2+) and H(2)O(2). The overall process of iron deposition and detoxification by LiDps is described by the following equations. For ferroxidation, Fe(2+) + Dps(Z)--> [(Fe(2+))-Dps](Z+1) + H(+) (Fe(2+) binding) and [(Fe(2+))-Dps](Z+1) + Fe(2+) + H(2)O(2) --> [(Fe(3+))(2)(O)(2)-Dps](Z+1) + 2H(+) (Fe(2+) oxidation/hydrolysis). For mineralization, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH((core)) + 4H(+) (Fe(2+) oxidation/hydrolysis). These reactions occur in place of undesirable odd-electron redox processes that produce hydroxyl radical.     

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.     

Metal Binding at the Deinococcus radiodurans Dps-1 N-Terminal Metal Site Controls Dodecameric Assembly and DNA Binding

The prokaryotic DNA protection during starvation (Dps) proteins typically protect macromolecules against damaging agents via physical association with DNA and by oxidizing and sequestering iron. However, Deinococcus radiodurans Dps-1, which binds DNA with high affinity, fails to protect DNA against hydroxyl radicals due to iron leakage from the core, raising the question of how (?)OH-mediated damage to Dps-1-bound DNA is avoided. As shown here, Mn(II) inhibits ferroxidase activity, suggesting that ferroxidation may be prevented in vivo as D. radiodurans accumulates a high ratio of Mn:Fe. Dps-1 has an N-terminal extension with a unique metal-binding site, an extension that has been proposed to be important for DNA binding and dodecameric assembly. Electrophoretic mobility shift assays show that Mn(II) restores DNA binding to bipyridyl-treated Dps-1, whereas Fe(II) fails to do so in the presence of H(2)O(2), thus preventing DNA binding under conditions of ongoing ferroxidase activity. We also show that disruption of the N-terminal metal site leads to a significant reduction in DNA binding and to compromised oligomeric assembly, with the mutant protein assembling into a hexamer in the presence of divalent metal. We propose that securing the N-terminal loop by metal binding is required to initiate dodecameric assembly by contacting the neighboring dimer and that the absence of such optimal contacts results in formation of a hexameric assembly intermediate in which three dimers associate about one of the 3-fold axes. Once dodecameric Dps-1 is assembled, metal binding no longer affects oligomeric state; instead, differential metal binding controls DNA interaction under conditions of oxidative stress.     

Overexpression and characterization of an iron storage and DNA-binding Dps protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N(2)-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps(T. erythraeurm) protein (Dps(tery)) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps(tery), and we found that Dps(tery), like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps(tery) binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps(tery) indicated that it has an iron core that resembles that of horse spleen ferritin.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Dps protects cells against multiple stresses during stationary phase

Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli. Dps homologs are found throughout the bacteria and in at least one archaeal species. Dps has been shown to protect cells from oxidative stress during exponential-phase growth. During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal. We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells. We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock. The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression.     

Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d(5) configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, with t(12) of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.     

The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni

We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H(2)O(2) or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.     

Paired Bacillus anthracis Dps (mini-ferritin) have different reactivities with peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.     

Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by site-directed mutagenesis in Streptococcus suis

H(2)O(2) is an unavoidable cytotoxic by-product of aerobic life. Dpr, a recently discovered member of the Dps protein family, provides a means for catalase-negative bacteria to tolerate H(2)O(2). Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H(2)O(2) + Fe(2+) --> (.)OH + (-)OH + Fe(3+)). We explored the in vivo function of Dpr in the catalase- and NADH peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive ( approximately 10(6)-fold) to H(2)O(2), (ii) Dpr incorporated iron in vivo, (iii) a putative ferroxidase center was present in Dpr, (iv) single amino acid substitutions D74A or E78A to the putative ferroxidase center abolished the in vivo iron incorporation, and (v) the H(2)O(2) hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the H(2)O(2) resistance is mediated by Dpr in vivo by its iron binding activity.     

Differential DNA binding and protection by dimeric and dodecameric forms of the ferritin homolog Dps from Deinococcus radiodurans

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D.radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(+/-0.1) degrees C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca(2+) (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry.     

Dps biomineralizing proteins: multifunctional architects of nature

Dps proteins are the structural relatives of bacterioferritins and ferritins ubiquitously present in the bacterial and archaeal kingdoms. The ball-shaped enzymes play important roles in the detoxification of ROS (reactive oxygen species), in iron scavenging to prevent Fenton reactions and in the mechanical protection of DNA. Detoxification of ROS and iron chaperoning represent the most archetypical functions of dodecameric Dps enzymes. Recent crystallographic studies of these dodecameric complexes have unravelled species-dependent mechanisms of iron uptake into the hollow spheres. Subsequent functions in iron oxidation at ferroxidase centres are highly conserved among bacteria. Final nucleation of iron as iron oxide nanoparticles has been demonstrated to originate at acidic residues located on the inner surface. Some Dps enzymes are also implicated in newly observed catalytic functions related to the formation of molecules playing roles in bacterium-host cell communication. Most recently, Dps complexes are attracting attention in semiconductor science as biomimetic tools for the technical production of the smallest metal-based quantum nanodots used in nanotechnological approaches, such as memory storage or solar cell development.