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Biomedical subjects

P R Shewry

Publications and source records attributed to P R Shewry.

At least 37 records · Page 2Linked to original sources
PubMed2000

Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.

Fatty Acid Desaturases↗
PubMed2000

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein.

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.

Amino Acid Sequence↗
PubMed2000

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.

Amino Acid Sequence↗
PubMed2000

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

2S Albumins, Plant↗
PubMed2000

Elastomeric proteins: biological roles, structures and mechanisms.

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain beta-turns as a structural motif within the elastomeric domains.

Amino Acid Sequence↗
PubMed2000

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli.

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography. The expressed protein exhibited electrophoretic, immunochemical, and surface hydrophobicity properties similar to those of native amarantin from amaranth seed. Also, the recombinant protein was refolded in vitro using two different methods.

Chromatography, High Pressure Liquid↗
PubMed2000

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers.

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.

Amino Acid Sequence↗
PubMed2000

Genetically modified crops: methodology, benefits, regulation and public concerns.

The genetic modification of crop plants from the methodology involved in their production through to the current debate on their use in agriculture are reviewed. Techniques for plant transformation by Agrobacterium tumefaciens and particle bombardment, and for the selection of transgenic plants using marker genes are described. The benefits of currently available genetically modified (GM) crops in reducing waste and agrochemical use in agriculture, and the potential of the technology for further crop improvement in the future are discussed. The legal requirements for containment of novel GM crops and the roles of relevant regulatory bodies in ensuring that GM crops and food are safe are summarized. Some of the major concerns of the general public regarding GM crops and food: segregation of GM and non-GM crops and cross-pollination between GM crops and wild species, the use of antibiotic resistance marker genes, the prevention of new allergens being introduced in to the food chain and the relative safety of GM and non-GM foods are considered. Finally, the current debate on the use of GM crops in agriculture and the need for the government, scientists and industry to persevere with the technology in the face of widespread hostility is studied.

Consumer Product Safety↗
PubMed2000

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans.

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.

Acetyltransferases↗
PubMed1999

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.

2S Albumins, Plant↗
PubMed1999

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds.

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin (Ki=100 pM). The crystal structure of this peptide in complex with bovine beta-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60-70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).

In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis. The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts. However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants. Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele. ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed. In maize, ZmeIF3A is encoded by one or two genomic sequences. This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex.

Amino Acid Sequence↗
PubMed1999

Identification of microphases in mixed alpha- and omega-gliadin protein films investigated by atomic force microscopy.

Pure and mixed films of alpha- and omega-gliadins were studied by tapping mode atomic force microscopy (AFM). The technique was sensitive to the chemistry of the surface properties of the films, allowing imaging of the mixed gliadin phases at different ratios. In addition to the study of the phases at the micrometer level, higher resolution images allowed visualization of the protein films at the molecular level. These studies may have relevance to the formation of phases in developing protein bodies in grain, where gliadins and glutenins are deposited together. It has been assumed that the protein bodies consist of a random network of proteins; these studies indicate that microphases could be present in protein bodies. The technique provides novel methods for studying mixed biopolymer systems.

Gliadin↗
PubMed1999

Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller).

Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OC-I delta 86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OC-I delta 86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs. The synthetic cysteine proteinase inhibitors E-64 and leupeptin also significantly reduced slug weight gain (by at least 40%) and digestive gland cysteine proteinase activity when administered in an artificial diet, indicating that their antimetabolic effects are due to direct inhibition of gut proteolytic activity. These results suggest that transgenic crop plants expressing phytocystatins could be used to suppress the growth rates of slug populations in the field.

Animals↗
PubMed1999

Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

Amino Acid Sequence↗
PubMed1998

Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.).

Oil bodies were isolated from mature seeds of sunflower (Helianthus annuus L.) and safflower (Carthamus tinctorius L.). Oil body preparations containing only oleosin proteins could be obtained from safflower seeds by salt-washing followed by centrifugation on discontinuous sucrose density gradients. However, it was necessary to treat sunflower oil bodies with urea to obtain preparations of similar purity. Incubation of the oil bodies with proteinases gave two fragments with molecular masses of 6 and 8 kDa which were protected from digestion. These fragments represented the hydrophobic domain of the oleosins, as determined by N-terminal sequencing. Intact and proteinase-treated oil bodies of both species were analysed by Fourier-transform infrared spectroscopy, as dry films and in aqueous medium, the spectra being compared with those obtained for pure oil samples in order to identify the bands resulting from the oleosin proteins and protected peptides. This investigation showed that the hydrophobic domain of the oleosins in intact oil bodies is predominantly alpha-helical in structure and that the conformation was not greatly affected by washing the oil bodies with urea during preparation.

Amino Acid Sequence↗
PubMed1998

Identification of a Caenorhabditis elegans Delta6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae.

We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Delta6-desaturase. A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae. This demonstrated that the protein encoded by the C. elegans cDNA was that of a fatty acid Delta6-desaturase, as determined by the accumulation of gamma-linolenic acid. The C. elegans Delta6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Delta6-desaturase. The C. elegans Delta6-desaturase mapped to cosmid W08D2, a region of chromosome III. This is the first example of a Delta6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.

Amino Acid Sequence↗