Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved.
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Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants of Scytalidium thermophilum catalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7Å, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haem b instead of the normal haem d. The H82A variant demonstrates low catalase and phenoloxidase activities (0.2 and 20% of those of recombinant wild-type catalase-phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haem d, showing that Asn155 is essential for catalysis but is not required for the conversion of haem b to haem d. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem. ©2013 International Union of Crystallography.
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Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an Arg-Gly-Asp cell adhesion motif and eight aspartic acid residues. In vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting the Clostridium thermocellum CipB cellulose binding domain (CBD) as a fusion partner protein.
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Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainlyα-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.
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Rational molecular design of self- assembling peptide-based materials that spontaneously form self-supporting hydrogels shows potential in many healthcare applications. Binary peptides based on complementary charged sequences are developed, and the use of biophysical analysis and cell-based studies highlights that the charged interactions can influence the properties of peptide materials and ultimately affect biomaterial applications.
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Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P₁₁-family of peptides includes, P₁₁-4 (QQRFEWEFEQQ) and the complementary peptides P₁₁-13 (EQEFEWEFEQE) and P₁₁-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P₁₁-4) or when mixed (P₁₁-13 and P₁₁-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage.
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Background: Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS) that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results: The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions: PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims. org/.© 2011 Troshin et al; licensee BioMed Central Ltd.
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This chapter focusses upon the current capacity for recombinant production of both structural proteins and peptides, including those capable of self-assembly. The prospects for large-scale production to deliver the quantities required for many engineering applications. Advantages and disadvantages of various protein expression systems will be presented with particular empthasis on microbial and plant-based system. Tools for assisting in the downstream processing and recovery of proteins and peptides will be considered. One of the applications of self-assembling peptides is for tissue engineering matrices. To date there are few examples of recombinant production of self-assembling peptides and these will be discussed. We will also consider examples of successful recombinant production of naturally occurring extracellular matrix proteins and of bioactive peptides as these offer important insights into the development of recombinant systems for future self-assembling peptide production.© 2009 Elsevier Inc. All rights reserved.
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Tranylcypromine (TCP), an amphetamine, is a reversible inhibitor of copper-containing amine oxidases. We have solved the structure of the complex of TCP with the amine oxidase from E. coli (ECAO) and shown that only the (+)-enantiomer of TCP binds. Kinetic studies on 2-phenylethylamine and TCP binding to wild-type ECAO and mutational variants fully support the model in which binding of the protonated amine is the first step in the catalytic cycle. Hydrazines are irreversible inhibitors of copper-containing amine oxidases. Binding of hydrazines leads to an adduct ('' Adduct 1 '') with a chromophore at 430 nm which converts at higher pH to another adduct ('' Adduct 2 '') with a chromophore at 520 nm. We have determined the structures of Adduct 1 and 2 for 2-hydrazinopyridine reacted with ECAO. It has been found that Adduct 1 corresponds to the hydrazone and azo tautomers whilst Adduct 2 corresponds to the azo tautomer coordinated to the active site copper. The implications of these results in developing more specific drugs are discussed.
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Information on PCR applications in genomics and proteomics will be expanded and integrated throughout the text.
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Galactose oxidase (GO; EC 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper and an amino acid-derived cofactor. The mechanism of this radical enzyme has been widely studied by structural, spectroscopic, kinetic and mutational approaches and there is a reasonable understanding of the catalytic mechanism and activation by oxidation to generate the radical cofactor that resides on Tyr-272, one of the copper ligands. Biogenesis of this cofactor involves the post-translational, autocatalytic formation of a thioether cross-link between the active-site residues Cys-228 and Tyr-272. This process is closely linked to a peptide bond cleavage event that releases the N-terminal 17-amino-acid pro-peptide. We have shown using pro-enzyme purified in copper-free conditions that mature oxidized GO can be formed by an autocatalytic process upon addition of copper and oxygen. Structural comparison of pro-GO (GO with the prosequence present) with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main chain position and some active-site-residue side chains differing significantly from their mature enzyme positions. These structural effects of the pro-peptide suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. Various models can be proposed to account for the formation of the thioether bond and oxidation to the radical state; however, the mechanism of prosequence cleavage remains unclear.
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Biotechnology offers sustainable solutions to the problem of plant parasitic nematode control. There are several possible approaches for developing transgenic plants with improved nematode resistance; these include anti-invasion and migration strategies, feeding-cell attenuation, and antinematode feeding and development strategies. The essential elements of an effective control strategy are (a) genes that encode an antinematode effector protein, peptide or interfering RNA and (b) promoters that direct a specific pattern of expression for that effector. This review summarizes information on effectors that act directly against the nematode as well as those aimed at disrupting the nematode feeding site. We discuss patterns of promoter activity that could deliver expression of these effectors in a restricted and directed manner. Societal opposition to the technology of GM-nematode control is also discussed.
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Plant nematodes are agricultural pests, the control of which relies on chemical nematicides and fumigants that are among the most toxic and environmentally damaging of all agrochemicals. New approaches to control, based on transgenic resistance, would provide important health and environmental benefits. In this chapter we consider briefly some targets for engineering nematode resistance and discuss the use of plant protease inhibitors as anti-feedants. This approach has provided plants that display good levels of resistance against a range of nematode species. To enhance this defence strategy further we are investigating the value of directed evolution to improve the characteristics of protease inhibitors. We describe the approaches of DNA shuffling and phage display that are being used to create and screen variant libraries in the search for inhibitors with improved features.
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This user friendly, practical guide to the basics of polymerase chain reaction is anintroduction to the essential tools needed for laboratory techniques.
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Redox interconversions between the GOase(semi) (Cu(II), Tyr) and tyrosyl radical containing GOase(ox) (Cu(II), Tyr(*)) oxidation states of the Cu-containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 have been studied. The inorganic complexes [Fe(CN)(6)](3)(-) (410 mV), [Co(phen)(3)](3+) (370 mV), [W(CN)(8)](3)(-) (530 mV), and [Co(dipic)(2)](-) (362 mV) (E degrees ' values vs NHE; dipic = 2,6-dicarboxylatopyridine) were used as oxidants for GOase(semi), and [Fe(CN)(6)](4)(-) and [Co(phen)(3)](2+) as reductants for GOase(ox). On oxidation of GOase(semi) a radical is generated at the coordinated phenolate of Tyr-272 to give GOase(ox). The one-electron reduction potential E degrees ' (25 degrees C) for the GOase(ox)/GOase(semi) couple varies with pH and is 400 mV vs NHE at pH 7.5, the smallest value so far observed for a tyrosyl radical. The reactions are very sensitive to pH, or more precisely to pK(a) values of GOase(semi) and GOase(ox), and the charge on the inorganic reagent. For example, with [Fe(CN)(6)](3)(-) as oxidant, the rate constant (25 degrees C)/M(-)(1) s(-)(1) of 0.16 x 10(3) (pH approximately 9.5) increases to 4.3 x 10(3) (pH approximately 5.5), while for [Co(phen)(3)](3+) a value of 4.9 x 10(3) (pH approximately 9.5) decreases to 0.04 x 10(3) (pH approximately 5.5), I = 0.100 M (NaCl). From the kinetics a single GOase(semi) acid dissociation process, pK(a) = 8.0 (average), has been confirmed by UV-vis spectrophotometric studies (7.9). The corresponding value for GOase(ox) is 6.7. No comparable kinetic or spectrophotometric pH dependences are observed with the Tyr495Phe variant, indicating the axial Tyr-495 as the site of protonation. Neutral CH(3)CO(2)H and HN(3) species bind at the substrate binding site of GOase(semi), thus mimicking the behavior of primary alcohols RCH(2)OH, the natural substrate of GOase. On coordination, loss of a proton occurs, and inhibition of the oxidation with [Fe(CN)(6)](3)(-) is observed.
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Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long propeptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-I Delta D86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.
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Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes. (C) 1997 Elsevier Science B.V.
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The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressive from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblottingin females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.
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Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.
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A pea seedling amine oxidase cDNA has been isolated and sequenced. A single long open reading frame has amino acid sequences corresponding to those determined from active site peptide (Janes, S.M., Palcic, M.M., Scaman, C.H., Smith, A.J., Brown, D.E., Dooley, D.M., Mure, M., and Klinman, J.P. (1992) Biochemistry 31, 12147-12154) and N-terminal sequencing experiments. The latter reveals the protein to have a 25-amino acid leader sequence with characteristics of a secretion signal peptide, as expected for this extracellular enzyme. Comparisons of the amino acid sequence of the mature pea enzyme (649 amino acids) with that of the mature lentil enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agrò, A. (1992) FEBS Lett. 301, 253-257) reveal important and unexpected differences particularly with regard to protein length. Sequencing of part of the lentil gene identified several frameshift differences within the coding region resulting in a mature lentil protein of exactly the same length, 649amino acids, as the pea enzyme. Multiple alignments of 10 copper amine oxidase sequences reveal 33 completely conserved residues of which 10 are found within 41 aligned residues at the C-terminal tails, the region missing from the original lentil sequence. One of only four conserved histidines is found in this region and may represent the third ligand to the copper. The pea enzyme contains around 3-4% carbohydrate as judged by deglycosylation experiments. We have also demonstrated by hybridization analysis that copper amine oxidase genes are present in a range of mono- and dicotyledonous plants.
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The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated. Inhibitory activities (Ki) of wild-type and variant forms of Oc-I against both papain and the C. elegans cysteine proteinase, gcp-1, were measured. For one variant, in which residue Asp86 was deleted (Oc-I deltaD86), the Ki was reduced by 13- to 14-fold. LD50 studies to test the effect of Oc-I and Oc-I delta D86 against C. elegans showed the relative median potency of Oc-I delta D86 to be 0.76 that of wild-type Oc-I. When expressed in tomato hairy roots both Oc-I and Oc-I delta D86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc-I deltaD86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.
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Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ).
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Cystatins are small protein inhibitors of cysteine proteinases. The relative importance of the N-terminal region of cystatins, and of a conserved glycine within this region, remains unclear despite several studies. It was found that deletion of the N-terminal 21 amino acids abolishes the inhibitory capacity of oryzacystatin-I. The importance of a conserved glycine residue (Gly10) was also examined by replacing it with 11 other amino acids. Three further glycine residues (Gly5, -6 and -11) in this N-terminal region of oryzacystatin-I were similarly mutated. Only those variants in which Gly10 was substituted show any significant change in inhibitory capacity compared with wild-type oryzacystatin-I. The inhibitory characteristics of hybrid cystatin molecules comprising regions of chicken egg white cystatin and oryzacystatin were also examined. It is suggested that in common with animal cystatins, the N-terminal region of the plant cystatin, oryzacystatin-I, and in particular the highly conserved Gly10 residue are important for effective inhibition of papain.
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PCR: A Practical Approach Volume 2 is not a revised version of PCR:A Practical Approach, but sets out to address some of the new and exciting applications of ...
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Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.
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The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.
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The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25 has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa. in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus. probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse aβ-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.
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The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.
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gdhA1 is a spontaneous mutant of Escherichia coli that causes complete loss of activity of the NADP-specific glutamate dehydrogenase (GDH) encoded by the gdhA gene. The gdhA1 mutational site has been identified by recombinational mapping, polymerase chain reaction (PCR) amplification and DNA sequencing, as an A to G transition at nucleotide 274 of the gdhA coding sequence, resulting in an amino acid change of lysine 92 to glutamic acid. The mutant enzyme forms hybrid hexamers with a wild-type GDH, providing a useful system for analysis of conformational integrity of mutational variants.
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Galactose oxidase is a copper-containing enzyme, which catalyses stereospecific oxidation of primary alcohols. The three-dimensional structure of the enzyme has been determined in this study by X-ray crystallography at high resolution. The molecule is almost entirely composed of beta-structures and consists of three domains. The arrangement of 28 beta-strands in the second domain is of particular interest, having seven four-stranded antiparallel beta-sheets with pseudo-sevenfold symmetry. The copper site has square-pyramidal coordination with two histidines, one tyrosine and one exogenous ligand at the equatorial sites and another tyrosine at the axial site. The most intriguing structural feature is a covalent bond between C epsilon 1 of Tyr-272, which is one of the equatorial ligands, and S gamma of Cys-228. This unexpected thioether bond, and Trp-290 stacked above it, strongly supports the presence of a tyrosine free radical in the enzyme as a 'built-in' secondary cofactor. Calculation of the molecular surface shows a small pocket at the copper site and suggests a substrate-binding model, which can explain the substrate specificity. A model for the catalytic mechanism, involving a tyrosine free radical and basic tryptophan, is also proposed.
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The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefaciens recA lacks the upstream 'SOS box', suggesting a different mechanism of regulation for this gene.
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The gaoA gene, encoding the secreted copper-containing enzyme galactose oxidase, has been isolated from the Deuteromycete fungus Dactylium dendroides. Degenerate oligonucleotide primers were designed from amino acid sequence data for use in the polymerase chain reaction. A 1.4-kilobase DNA fragment amplified from genomic DNA was used to screen a genomic library constructed in ZAP. A strongly hybridizing clone was rescued as a pBluescript derivative, pGAO9, by in vivo excision. The sequence of 3466 nucleotides of pGAO9 insert DNA was determined by progressively designing sequencing primers. The translation product of the single long open reading frame matches the available galactose oxidase peptide sequence data, which represents 42% of the residues in the protein. The mature enzyme has 639 residues, which have been assigned to a 1.7-A electron density map (Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J., Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991) Nature 350, 87-90). The gene lacks introns and encodes an mRNA of approximately 2.5 kilobases with three transcription initiation start points at least 324 nucleotides upstream of the translation start site. Multiple ATG codons are present between the transcription initiation region and the start of the mature protein; two in-frame ATGs could encode the initiating Met residue to give proteins with 89 or 41 residue N-terminal leader peptides. The shorter potential leader has N-terminal features characteristic of a secretion signal sequence and may also contain a pro-sequence processed by an enzyme specific for a monobasic (arginine) cleavage site, as proposed for other fungal genes. The codon bias of gaoA is characteristic of other filamentous fungal genes. No significant homologies exist between galactose oxidase and other protein sequences available in data bases.
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Peptide sequence data derived from a plant annexin, P34 [Smallwood, Keen&Bowles (1990) Biochem. J. 270, 157-161] was used to design amplimers for PCR. A unique fragment of 95 bp, amplified from tomato (Lycopersicon esculertum) genomic DNA, was used in Northern analyses and demonstrated a differential pattern of expression in vegetative tissues of tomato, potato (Solanum tuberosum) and barley (Hordeum vulgare). The tissue-specific abundance of the annexin transcript was found to correlate closely with abundance of annexin protein as revealed by their partial purification and analysis with antisera specific for annexins isolated from tomato suspension-culture cells.
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This volume and its companion together provide the first comprehensive guide to the latest molecular techniques as well as the established approaches to the ...
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This volume and its companion together provide the first comprehensive guide to the latest molecular techniques as well as the established approaches to the ...
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Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium dendroides. It is monomeric, with a relative molecular mass of 68,000, catalyses the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalysing a two-electron transfer reaction during the oxidation of primary alcohols to corresponding aldehydes. Recent evidence arguing against a Cu(III)-Cu(I) couple implies the existence of a second redox-active site proposed to involve pyrroloquinoline quinone or a tyrosine radical. We now report the crystal structure of galactose oxidase at 1.7 A resolution. This reveals a unique structural feature at the copper site with a novel thioether bond linking Cys 228 and Tyr 272 in a stacking interaction with Trp 290. We propose that these molecular components stabilize the protein free-radical species essential for catalysis and thus provide a 'built-in' secondary cofactor. This feature may represent a new mechanism for mediating electron transfer in metalloenzymes in the absence of exogenous cofactors.
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A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.
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This book provides a general introduction to PCR for researchers new to the area.
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The gltBY locus of E. coli, which codes for the two subunits of pyridine nucleotide dependent glutamate synthase, was used as a probe to detect homologues in genomic DNA of Synechococcus PCC6301, a unicellular cyanobacterium. Non-overlapping probes from gltB detected a single homologue with extensive homology, however gltY probes do not detect a strong homologue. The possibility that the gltB homologue encodes a ferredoxin-dependent glutamate synthase of the type found in cyanobacteria, algae and plants is discussed.
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We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.
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Pullulanase from Klebsiella pneumoniae strain FG9 has an unusual N-terminal amino acid sequence that includes six repeats of the tripeptide Gly-X-Pro. This type of sequence is characteristic of animal collagens and collagen-like proteins which form triple helical structures. We have investigated the molecular organization of this bacterial pullulanase isolated from the cell surface of Escherichia coli cells that carry the cloned FG9 pulA (pullulanase encoding) gene. Non-denaturing polyacrylamide gel analysis shows that pullulanase exists as higher order, apparently homogeneous, structures. We have used highly purified bacterial collagenase to probe the role of the collagen-like region and we demonstrate that this feature is essential for non-covalent association of pullulanase homotrimers. In addition we show collagenase-specific release of cell-bound pullulanase.
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A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.
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