细菌脲酶蛋白复合物UreABC的表达与纯化

细菌脲酶能分解尿素为氨,在瘤胃尿素氮代谢中发挥重要作用.为了表达纯化并研究蛋白脲酶复合物UreABC,通过PCR扩增脲酶基因簇的结构基因ureA、ureB、ureC,将ureB构建在含有N端His标签的pet28a+载体,ureA、ureC基因共同构建在含有N端His标签的表达载体pETDuet-1,经酶切及测序鉴定得...     

细菌脲酶蛋白复合物及其活化机制

脲酶能够催化尿素分解生成氨,在农业和医学领域中具有重要的意义。细菌脲酶蛋白包括结构蛋白(UreA、UreB和UreC)和辅助蛋白(UreD/UreH、UreE、UreF和UreG),它们在脲酶活化过程中各自具有独特的作用,结构蛋白形成脲酶活性中心,而辅助蛋白主要负责镍离子的传递。文中综述了细菌脲酶蛋白复合物的结构和功能,以及各蛋白之间如何相互作用完成其活化过程,以期为脲酶活性调控研究及脲酶抑制剂开发等提供理论指导。     

A putative β-glucanase pseudogene behind the potato GBSS gene

We identified an open reading frame (ORF) which is located closely behind the gene encoding granulebound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The deduced protein shows homology with all known isoforms of plant -1,3-glucanases and -1,3-1,4-glucanases. Although the DNA sequence is unique in potato and tomato (Lycopersicon esculentum L.), no expression of the gene was found in these species. Taken together with the unusual codon usage and length of the predicted protein, this sequence could be a pseudogene.     

Coptisine-induced inhibition of Helicobacter pylori: elucidation of specific mechanisms by probing urease active site and its maturation process

In this study, we examined the anti-Helicobactor pylori effects of the main protoberberine-type alkaloids in Rhizoma Coptidis. Coptisine exerted varying antibacterial and bactericidal effects against three standard H. pylori strains and eleven clinical isolates, including four drug-resistant strains, with minimum inhibitory concentrations ranging from 25 to 50?μg/mL and minimal bactericidal concentrations ranging from 37.5 to 125?μg/mL. Coptisine’s anti-H. pylori effects derived from specific inhibition of urease in vivo. In vitro, coptisine inactivated urease in a concentration-dependent manner through slow-binding inhibition and involved binding to the urease active site sulfhydryl group. Coptisine inhibition of H. pylori urease (HPU) was mixed type, while inhibition of jack bean urease was non-competitive. Importantly, coptisine also inhibited HPU by binding to its nickel metallocentre. Besides, coptisine interfered with urease maturation by inhibiting activity of prototypical urease accessory protein UreG and formation of UreG dimers and by promoting dissociation of nickel from UreG dimers. These findings demonstrate that coptisine inhibits urease activity by targeting its active site and inhibiting its maturation, thereby effectively inhibiting H. pylori. Coptisine may thus be an effective anti-H. pylori agent.     

Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato

We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3 non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.     

Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K_m = 67 mm, k_cat = 490 s⁻¹). The activity depended on the presence of manganese (K_d = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.Nitrogen (N) availability often limits plant performance in natural ecosystems (Vitousek and Howarth, 1991), causing a selective pressure to optimize the use of N resources. This ecophysiological selection has even led to a reduction of the N content of plant proteins in comparison with animal orthologs (Elser et al., 2006). Because N is a limiting resource, plants do not only require efficient N uptake mechanisms but also possess enzymatic pathways for N remobilization.Arg is the most important single metabolite for N storage in plant seeds. In a survey of 379 plant species, Arg N accounted on average for 17.3% of total seed N (Vanetten et al., 1967). In several rice (Oryza sativa) varieties, values ranging from 16.1% to 17.1% were measured (Mosse et al., 1988). To access the N stored in the guanidinium group of Arg, it must first be hydrolyzed by mitochondrial arginase to Orn and urea. Urea leaves the mitochondria and is hydrolyzed by urease in the cytosol, releasing ammonia, which is reassimilated into amino acids by the combined action of Gln synthetase and Glu synthase.Urea not only originates from Arg breakdown but may also be taken up from the environment by urea transporters (Kojima et al., 2007; Wang et al., 2008). Therefore, urease is involved in N remobilization as well as in primary N assimilation. Plant ureases and arginases are housekeeping enzymes found in many if not all plant species (Witte and Medina-Escobar, 2001; Brownfield et al., 2008). Urease is a nickel metalloenzyme that in Arabidopsis (Arabidopsis thaliana) requires three urease accessory proteins (UAPs; AtUreD, AtUreF, and AtUreG) for activation (Witte et al., 2005a). Studies in bacteria demonstrated that UAPs form a complex with apo-urease and are required for posttranslational Lys carboxylation of apo-urease and the subsequent incorporation of two nickel ions into the active center. After activation, the UAPs dissociate from urease. The exact molecular function of each accessory protein in this process is not yet understood (Carter et al., 2009). Like urease, arginase is a metalloenzyme. It is best activated by manganese (Carvajal et al., 1996; Hwang et al., 2001), not requiring accessory proteins for activation.Urea plays an important role in agriculture because it is the most used N fertilizer worldwide (http://www.fertilizer.org/ifa), intensively employed in Asia for the cultivation of rice. Urea N partly reaches the plant as ammonium or nitrate because the fertilizer is already degraded in the environment by microbial ureases and may then be subject to nitrification. Alternatively, plants are capable of taking up urea from fertilization directly and assimilate its N (Kojima et al., 2007; Wang et al., 2008). Although rice is a major crop plant and rice production is heavily dependent on urea fertilization, the enzymes and the corresponding genes involved in rice urea metabolism have not yet been investigated. In this study, we identified the genes and cloned the corresponding cDNAs coding for rice arginase, urease, and the UAPs UreD, UreF, and UreG. The functionality of the corresponding proteins was demonstrated and biochemical parameters were determined. The general gene and protein structure of plant UreD and UreF were investigated and a direct interaction of rice UreD with apo-urease was discovered, leading to a refinement of the mechanistic view of plant urease activation. In physiological experiments, rice urease and arginase activities showed no significant response to different N-fertilizing regimes, while the amino acid composition in urea-grown plants was strongly imbalanced, indicating that urea N disturbs plant metabolism downstream of N assimilation.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.Identification of virulence factors is vitally important to an understanding of the pathogenesis of Edwardsiella ictaluri and to the development of methods for controlling the spread of disease. Although the pathogenesis of E. ictaluri was reviewed in 1993 (28, 31), recent reports demonstrated that E. ictaluri is a facultative intracellular pathogen (3) and that a type III secretion system is required for intracellular survival and replication within channel catfish head kidney-derived macrophages (HKDM) (30). Using signature-tagged mutagenesis (STM) in an immersion challenge model for E. ictaluri, Thune et al. (30) identified 50 transconjugants carrying transposon insertions in genes required for survival and replication in the channel catfish host. Two of those mutants had insertions in genes encoding homologs of UreG and UreF, proteins that are essential for the production of an active urease enzyme in other bacteria (6, 10, 14, 26). UreG is a GTP-binding accessory protein that functions in energy-dependent assembly of the urease holoenzyme (19), while UreF is a urease accessory protein that functions in the generation or delivery of carbon dioxide to the urease metallocenter assembly site (19). Both the ureG and ureF mutant strains were further characterized in a competitive challenge with the wild-type (WT) parental strain and were confirmed to be significantly attenuated (30). The identification of two mutants with insertions in urease-associated genes suggests an important role for urease activity in E. ictaluri pathogenesis, despite the fact that E. ictaluri is urease negative in standard biochemical tests. Consequently, the objectives of this study are to characterize the E. ictaluri urease pathogenicity island (PAI), to evaluate conditions for E. ictaluri urease activity, and to establish a possible role for urease in E. ictaluri pathogenesis.     

Soybean genes involved in nickel insertion into urease

In soybean, mutation in the Eu2 or in the Eu3 gene eliminates the activities, but not the proteins, of the embryo-specific and the ubiquitous ureases, encoded by Eu1 and Eu4, respectively. This pleiotropic urease-negative phenotype is consistent with accessory gene functions encoded by Eu2 and Eu3, i.e. correct insertion of Ni into the metallocentre of each urease. To test an accessory gene function an examination was made of segregation of alleles at Eu2 and Eu3 with segregation of an RFLP revealed by a plant homologue of the bacterial urease accessory gene, ureG. The eu3-e1/eu3-e1 mutant, which has a urease activity-null phenotype, lacked a 1.4 kb EcoRV genomic fragment found in progenitor cultivar Williams, cv Williams 82 and in two mutants at the Eu2 locus, eu2/eu2 and EN24, the latter described here for the first time. The lack of the 1.4 kb band segregated with eu3-e1 in a cross of eu3-e1/eu3-e1 x EN24. The second approach was to attempt partial correction of the urease-negative trait by Ni supplementation in vitro. First a small, reproducible stimulation of activity in mixed extracts of mutants which complement genetically, namely {eu2/eu2 plus eu3-e1/eu3-e1} and {EN24 plus eu3-e1/eu3-e1} was observed. Activation proceeded for several hours in these extracts containing endogenous Ni. In mixed extracts from Ni-free embryos, activation was dependent on added Ni; Ni had no effect on individual mutant extracts. By genetic and biochemical criteria the ubiquitous urease was the sole or major species activated, an activation which approached 10% normal activity.Key words: Nickel, urease, soybean, UreG, UreE.      

Ynt is the primary nickel import system used by Proteus mirabilis and specifically contributes to fitness by supplying nickel for urease activity

Proteus mirabilis is a Gram-negative uropathogen and frequent cause of catheter-associated urinary tract infection (CAUTI). One important virulence factor is its urease enzyme, which requires nickel to be catalytically active. It is, therefore, hypothesized that nickel import is critical for P. mirabilis urease activity and pathogenesis during infection. P. mirabilis strain HI4320 encodes two putative nickel import systems, designated Nik and Ynt. By disrupting the substrate-binding proteins from each import system (nikA and yntA), we show that Ynt is the primary nickel importer, while Nik only compensates for loss of Ynt at high nickel concentrations. We further demonstrate that these are the only binding proteins capable of importing nickel for incorporation into the urease enzyme. Loss of either nickel-binding protein results in a significant fitness defect in a murine model of CAUTI, but YntA is more crucial as the yntA mutant was significantly outcompeted by the nikA mutant. Furthermore, despite the importance of nickel transport for hydrogenase activity, the sole contribution of yntA and nikA to virulence is due to their role in urease activity, as neither mutant exhibited a fitness defect when disrupted in a urease-negative background.     

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis -1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.     

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

Helicobacter pylori colonizes the human gastric mucosa and this can lead to chronic gastritis, peptic and duodenal ulcers, and even gastric cancers. The bacterium colonizes over one-half of the worlds population. Nickel plays a major role in the bacteriums colonization and persistence attributes as two nickel enzyme sinks obligately contain the metal. Urease accounts for up to 10% of the total cellular protein made and is required for initial colonization processes, and the hydrogen oxidizing hydrogenase provides the bacterium a high-energy substrate yielding low potential electrons for energy generation. A battery of accessory proteins are needed for maturation or activation of each of the apoenzymes. These include Ni-chaperones and GTPases, some of which are unique to each Ni-enzyme and others that are individually required for maturation of both the Ni-enzymes. H. pylori’s need for some conventional hydrogenase maturation proteins playing roles in urease maturation may have to do with the poor nickel-sequestering ability of the UreE urease maturation protein compared to other systems. H. pylori also possesses a NixA nickel specific permease, a nickel dependent regulator (NikR), a recently identified nickel efflux system (CznABC), and a histidine-rich heat shock protein, HspA. Based on mutant analysis approaches all these proteins have roles in nickel homeostasis, in urease expression, and in host colonization. The His-rich putative nickel storage proteins Hpn and Hpn-like play roles in nickel detoxification and may influence the levels of Ni-activated urease that can be achieved.     

Effect of the urease accessory genes on activation of the Helicobacter pylori urease apoprotein

The roles that accessory gene products play in activating the Helicobacter pylori urease apoprotein were examined. The activity of the urease apoprotein increased in the following order when it was expressed with the accessory genes: ureG相似文献   

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.