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Cloning of the virulence regulatory (vir) locus of Bordetella pertussis and its expression in B. bronchiseptica 总被引:2,自引:0,他引:2
The gene coding for a thermostable alpha-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulfurogenes carrying the alpha-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium. 相似文献
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目的建立能稳定分泌抗兔支气管败血波氏杆菌(Bb)的单克隆抗体杂交瘤细胞株,为今后进一步建立该菌的免疫检测技术奠定基础。方法以Bb分离株BLJ05的灭活菌液为免疫原,腹腔免疫BALB/c小鼠,采用常规杂交瘤技术制备Bb单克隆抗体(McAb),用间接ELISA、Western-blot等方法对McAb特性进行鉴定。结果获得两株能稳定分泌抗Bb单克隆抗体的杂交瘤细胞株,分别命名为A7D5和D6B2,其小鼠腹水抗体效价分别为1∶409600和1∶102400;且不与兔大肠杆菌、多杀性巴氏杆菌、产气荚膜梭菌等兔的常见病原菌反应,特异性强。两株单抗亲和力实验表明A7D5亲和力略高于D6B2。ELISA相加试验表明它们针对相同的抗原表位。结论成功建立了两株能稳定分泌抗兔支气管败血波氏杆菌单克隆抗体的杂交瘤细胞株,效价高、特异性强,为今后建立该菌的免疫检测技术建立奠定了基础。 相似文献
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Jacek Bajor Karolina L. Tkaczuk Maksymilian Chruszcz Hutton Chapman Olga Kagan Alexei Savchenko Wladek Minor 《Journal of structural and functional genomics》2014,15(2):73-81
We report a 2.0 Å structure of the CAE31940 protein, a proteobacterial NMT1/THI5-like domain-containing protein. We also discuss the primary and tertiary structure similarity with its homologs. The highly conserved FGGXMP motif was identified in CAE31940, which corresponds to the GCCCX motif located in the vicinity of the active center characteristic for THi5-like proteins found in yeast. This suggests that the FGGXMP motif may be a unique hallmark of proteobacterial NMT1/THI5-like proteins. 相似文献
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M N Rozinov Iu L Shumakov A A Da?n T A Gol'tsma?er V N Gershanovich 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1988,(4):90-93
The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Cytosolic D-ribulose-5-phosphate 3-epimerase from rice was crystallized after EDTA treatment and structurally elucidated by X-ray diffraction to 1.9A resolution. A prominent Zn(2+) site at the active center was established in a soaking experiment. The structure was compared with that of the EDTA-treated crystalline enzyme from the chloroplasts of potato plant leaves showing some structural differences, in particular the "closed" state of a strongly conserved mobile loop covering the substrate at its putative binding site. The previous proposal for the active center was confirmed and the most likely substrate binding position and conformation was derived from the locations of the bound zinc and sulfate ions and of three water molecules. Assuming that the bound zinc ion is an integral part of the enzyme, a reaction mechanism involving a well-stabilized cis-enediolate intermediate is suggested. 相似文献
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Halak S Lehtiö L Basta T Bürger S Contzen M Stolz A Goldman A 《The FEBS journal》2006,273(22):5169-5182
3-carboxy-cis,cis-muconate lactonizing enzymes participate in the protocatechuate branch of the 3-oxoadipate pathway of various aerobic bacteria. The gene encoding a 3-carboxy-cis,cis-muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3-carboxy-cis,cis-muconate lactonizing enzyme of 353 amino acids - significantly smaller than all previously studied 3-carboxy-cis,cis-muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3-carboxy-cis,cis-muconate but also 3-sulfomuconate. ArCMLE1 was purified as a His-tagged enzyme variant, and the basic catalytic constants for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate were determined. In contrast, Agrobacterium tumefaciens 3-carboxy-cis,cis-muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3-sulfomuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 A resolution. Consistent with the sequence, it showed that the C-terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3-carboxy-cis,cis-muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424-10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site. 相似文献
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The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules. The second product, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, is recycled in the biosynthetic pathway by 6,7-dimethyl-8-ribityllumazine synthase. This article will review structures and reaction mechanisms of riboflavin synthases and related proteins up to 2007 and 122 references are cited. 相似文献
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Malic enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme in a quaternary complex with NAD+, Mn++ and oxalate has been determined at 2.2 A resolution. The structures of the quaternary complex with NAD+, Mg++, tartronate or ketomalonate have been determined at 2.6 A resolution. The structures show the enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of malic enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control. 相似文献
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Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization. 相似文献
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Huang J Liang Z Yang B Tian H Ma J Zhang H 《The Journal of biological chemistry》2007,282(46):33632-33640
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies. 相似文献
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4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme 总被引:1,自引:0,他引:1
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4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower Vmax and a four-fold higher Km value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components. 相似文献
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Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus
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Cytochrome c oxidase is a respiratory enzyme catalysing the energy-conserving reduction of molecular oxygen to water. The crystal structure of the ba(3)-cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 A resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure-based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from Cu(A) to Cu(B). Specific features of the ba(3)-oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O(2-), OH(-) or H(2)O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified. 相似文献
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Ganichkin OM Xu XM Carlson BA Mix H Hatfield DL Gladyshev VN Wahl MC 《The Journal of biological chemistry》2008,283(9):5849-5865
In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA [Ser]Sec into selenocysteyl-tRNA [Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA [Ser]Sec synthesis activity, and determined its crystal structure at 1.65 A resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free alpha-carboxyl group, suggesting why SecS does not act on free O-phospho-l-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the gamma-phosphate moiety of O-phospho-l-seryl-tRNA [Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA [Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme. 相似文献
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Stogios PJ Shakya T Evdokimova E Savchenko A Wright GD 《The Journal of biological chemistry》2011,286(3):1966-1975
The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme. 相似文献