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1.
H Alexander S Alexander F Heffron T M Fieser B N Hay E D Getzoff J A Tainer R A Lerner 《Gene》1991,99(2):151-156
The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein. 相似文献
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Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions. 总被引:8,自引:7,他引:1
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Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides. 相似文献
4.
Divergence of the aerobactin iron uptake systems encoded by plasmids pColV-K30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1. 总被引:8,自引:1,他引:7
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Although the aerobactin-mediated iron uptake system has been characterized genetically in Escherichia coli, the siderophore aerobactin was chemically characterized after purification from culture supernatants of Aerobacter aerogenes 62-1, a member of the Klebsielleae. We have cloned and mapped the genes encoding the aerobactin system genes of A. aerogenes 62-1 and begun characterization of the relevant proteins and enzymatic activities of this plasmid-mediated aerobactin system. Published chemical data indicate that the siderophore aerobactin of E. coli is the same molecule as the aerobactin of Aerobacter aerogenes 62-1, but we have found that both the genes and the complement of proteins making up the biosynthetic enzymes in the two systems have diverged. In contrast, the outer membrane receptors for ferric aerobactin of the two systems showed immunologic cross-reactivity, were of the same molecular size (74 kilodaltons), and were encoded by homologous DNA sequences. 相似文献
5.
Subcloning of the cloacin DF13/aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake. 总被引:9,自引:6,他引:3
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W J Krone J Luirink G Koningstein B Oudega F K de Graaf 《Journal of bacteriology》1983,156(2):945-948
A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin. 相似文献
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Willy J.A. Krone Freek Stegehuis Gregory Koningstein Carla van Doorn Bert Roosendaal Frits K. de Graaf Bauke Oudega 《FEMS microbiology letters》1985,26(2):153-161
Abstract The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent Mr values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 108 M−1 and 1.0 × 109 M−1 , respectively, were found.
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an Mr of 77 345. 相似文献
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M
8.
Maqbool QU Johri S Rasool S Riyaz-ul-Hassan S Verma V Nargotra A Koul S Qazi GN 《Journal of biotechnology》2006,125(1):1-10
An isolated strain of Bacillus subtilis identified by 16S rDNA sequence analysis produces an enantioselective ester hydrolase. Whole cells of B. subtilis (RRL BB1) and enzyme derived from it was capable of enantioselective hydrolysis of several racemates including drug intermediates with moderate to high enantioselectivity as already reported by us. In this communication, we describe cloning of the gene encoding the enantioselective esterase designated as estBB1. The primary structure of the enzyme determined from the nucleotide sequence indicated that esterase estBB1 has Mw approximately 52kDa and pI approximately 5.2 and belongs to the family of type B carboxylesterases with 50-60% similarity at amino acid level. Alignment studies of sequences of the estBB1 and Pnb esterase 56C8 from B. subtilis showed that estBB1 has an alpha/beta hydrolase fold with catalytic triad formed by Ser190, Glu305 and His394 at active site and Ser190 is located in the conserved motif -G-X-S-X-G-. 相似文献
9.
Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed. 相似文献
10.
Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis. 总被引:15,自引:0,他引:15
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We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes. 相似文献
11.
Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. 总被引:7,自引:0,他引:7
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We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments. 相似文献
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Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2) 总被引:10,自引:0,他引:10
Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general. 相似文献
13.
Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed. 相似文献
14.
Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The Km values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern–Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M?1 and 0.56 M?1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. 相似文献
15.
Dell'Angelica EC Aguilar RC Wolins N Hazelwood S Gahl WA Bonifacino JS 《The Journal of biological chemistry》2000,275(2):1300-1306
Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of approximately 80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was approximately 6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the beta3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1 gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex. 相似文献
16.
HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria. 相似文献
17.
Angela Ostuni Maria Antonietta Castiglione Morelli Flavia Cuviello Alfonso Bavoso Faustino Bisaccia 《生物化学与生物物理学报:生物膜》2019,1861(2):380-386
ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E205-G279 contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. 1H NMR spectroscopy identified three helical regions separated by more flexible regions. 相似文献
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A novel cell surface-anchored cellulose-binding protein encoded by the sca gene cluster of Ruminococcus flavefaciens
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Rincon MT Cepeljnik T Martin JC Barak Y Lamed R Bayer EA Flint HJ 《Journal of bacteriology》2007,189(13):4774-4783
Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein ( approximately 130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species. 相似文献
20.
Miller DJ Shuvalova L Evdokimova E Savchenko A Yakunin AF Anderson WF 《Protein science : a publication of the Protein Society》2007,16(7):1338-1348
Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis-p-nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 A resolution. YfcE has a beta-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique. 相似文献