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G Williamson

Publications and source records attributed to G Williamson.

At least 91 records · Page 5Linked to original sources
PubMed1997

Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.

Aspergillus niger↗
PubMed1997

Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin.

BACKGROUND: Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. RESULTS: The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. CONCLUSIONS: The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules.

Aspergillus niger↗
PubMed1997

Effect of glucosinolate breakdown products on beta-naphthoflavone-induced expression of human cytochrome P450 1A1 via the Ah receptor in Hep G2 cells.

The ability of glucosinolates, the main bioactive components of brassica vegetables, to influence beta-naphthoflavone-induced transcription of human cytochrome P450 1A1 was examined. Human Hep G2 cells, which contain a functional Ah receptor, were transiently transfected with a reporter construct containing the entire promoter sequence of the human CYP1A1 gene. Breakdown products of four of the glucosinolates tested (prop-2-enyl, 3-methylsulphinylpropyl, 2-hydroxybut-3-enyl and p-hydroxybenzyl), at a concentration as low as 1 microM, reduced the level of beta-naphthoflavone-induced transcription of CYP1A1. It is already known that certain glucosinolate breakdown products can induce phase II enzymes and inhibit the catalytic activity of some cytochrome P450 isoenzymes. This study demonstrates that certain alkyl and aromatic glucosinolates may also influence cytochrome P450 1A1 transcription.

Carcinoma, Hepatocellular↗
PubMed1997

Chemical and thermal stability of ferulic acid esterase-III from Aspergillus niger.

The stability of ferulic acid esterase III (FAE-III) from Aspergillus niger was examined using chemical and thermal denaturation. Thermal denaturation was irreversible and the loss of activity was dependent on pH. At 60 degrees C and pH 6.0, the rate constant of unfolding was 0.76 10(-3)/s, and the change in free energy of irreversible inactivation, deltaG*, was 101.9 kJ/mol. Sinapic acid, a product of the reaction of methyl sinapate with FAE-III, reduced the rate of unfolding (0.66 10(-3)/s at 0.1 mM sinapic acid). Chemical denaturation was performed using guanidine hydrochloride. FAE-III was very sensitive to this denaturant, and the midpoint of unfolding was 1.38 M guanidine hydrochloride at 30 degrees C, pH 6.0. The stability of FAE-III is compared to other enzymes.

Aspergillus niger↗
PubMed1997

Mechanism of action of dietary chemoprotective agents in rat liver: induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism.

A range of potential chemoprotective agents, most of them natural dietary constituents, has been examined for ability to modulate both phase I (cytochrome P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A, 4A) and phase II drug metabolizing enzymes (glutathione S-transferases, in particular subunits Yc2 and P, aflatoxin B1-aldehyde reductase and quinone reductase) in rat liver. In addition to assays of total enzyme activity and Western blots for individual isozymes, the ability of microsomes to metabolize aflatoxin B1, and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to GSH and to produce AFB1-dialcohol, were measured. Induction of gamma-glutamyl transpeptidase activity was examined by histochemistry. Differing patterns of induction were observed, reflecting differences in the control of expression of the individual enzymes studied. Of the compounds examined, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol and phenethyl isothiocyanate were the most potent bifunctional agents (inducing both phase I and II activities). Oltipraz, while only weakly inducing CYP1A2 and 2B1/2, was a potent inducer of phase II enzymes. Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce phase II enzymes. 4-Methyl catechol, alpha-tocopherol and red wine decreased certain phase I enzyme activities, while inducing total GST activity. Butylated hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz, phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if administered prior to AFB1. While these studies indicate the relative contributions of phase I and II metabolism in the overall protective effect in rat, care should be taken that a similar balance is achieved in man, and that relevant enzymes or iso forms are induced.

Aflatoxin B1↗
PubMed1997

An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.

Aspergillus niger↗
PubMed1997

The faeA genes from Aspergillus niger and Aspergillus tubingensis encode ferulic acid esterases involved in degradation of complex cell wall polysaccharides.

We report the cloning and characterization of a gene encoding a ferulic acid esterase, faeA, from Aspergillus niger and Aspergillus tubingensis. The A. niger and A. tubingensis genes have a high degree of sequence identity and contain one conserved intron. The gene product, FAEA, was overexpressed in wild-type A. tubingensis and a protease-deficient A. niger mutant. Overexpression of both genes in wild-type A. tubingensis and an A. niger protease-deficient mutant showed that the A. tubingensis gene product is more sensitive to degradation than the equivalent gene product from A. niger. FAEA from A. niger was identical to A. niger FAE-III (C. B. Faulds and G. Williamson, Microbiology 140:779-787, 1994), as assessed by molecular mass, pH and temperature optima, pI, N-terminal sequence, and activity on methyl ferulate. The faeA gene was induced by growth on wheat arabinoxylan and sugar beet pectin, and its gene product (FAEA) released ferulic acid from wheat arabinoxylan. The rate of release was enhanced by the presence of a xylanase. FAEA also hydrolyzed smaller amounts of ferulic acid from sugar beet pectin, but the rate was hardly affected by addition of an endo-pectin lyase.

Amino Acid Sequence↗
PubMed1997

Antioxidant properties of the major polyphenolic compounds in broccoli.

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.

Antioxidants↗
PubMed1996

Solution structure of the granular starch binding domain of glucoamylase from Aspergillus niger by nuclear magnetic resonance spectroscopy.

The solution structure of the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger has been determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy and simulated annealing. A total of 1092 nuclear Overhauser enhancement-derived 1H-1H distance constraints, 137 dihedral constraints and 86 hydrogen bond constraints were incorporated into an X-PLOR simulated annealing and refinement protocol. The family of calculated structures shows a well defined beta-sheet structure consisting of one parallel and six antiparallel pairs of beta-strands which forms an open-sided beta-barrel. The root-mean-square deviation (rmsd) of 53 individual structures to the calculated average structure for the backbone atoms of residues excluding the N terminus and two mobile loops is 0.57(+/-0.10) A while the rmsd for backbone atoms in beta-strands is 0.45(+/-0.08) A. Structural features of the SBD in solution are compared to the X-ray crystal structure of a homologous domain of cyclodextrin glycosyltransferase (CGTase) in the free and bound forms. Titration studies with two ligands, maltoheptaose and beta-cyclodextrin, show the existence of two binding sites. Examination of the tertiary structures shows these two sites to be at one end of the molecule on opposite faces. The majority of residues showing the largest 1H and 15N chemical shift changes are located in loop regions. Many residues implicated in binding, based on these changes, are similar in location to previously identified binding site residues in the crystal structures of CGTase. Overall, the shift changes are small indicating that the SBD does not undergo large conformational changes upon ligand binding.

Aspergillus niger↗
PubMed1996

Transcriptional regulation of the human NAD(P)H:quinone oxidoreductase (NQO1) gene by monofunctional inducers.

The upstream region of the human NAD(P)H:quinone oxidoreductase (NQO1) gene contains a functional antioxidant responsive element (ARE) and an overlapping 12-O-tetradecanoyl-phorbol-13-acetate responsive element (TRE), with the sequence TGACTCAGCA. We show that the ARE (TGACNNNGCA) is required for induction by redox cycling phenolics (p-benzoquinone, catechol and hydroquinone), which are monofunctional inducers and induce NQO1 without the requirement for activation by cytochrome P-450. The TRE (TGACTCA) is involved only in basal expression. A plasmid containing overlapping ARE-TRE (TGACTCAGCA) sequences (-587 to -379) from the NAD(P)H:quinone oxidoreductase gene was transiently transfected into Hep G2 cells. In the absence of inducers, basal expression was 4-fold higher than in F9 cells (which lack AP-1 activity). Using subcloned oligonucleotides containing the ARE-TRE sequence (-473 to -440), the ARE sequence alone (TCA changed to GAC) and the TRE sequence alone (GC changed to TA), the basal level of expression was in the order: TRE > TRE-ARE > ARE in Hep G2 cells. Using F9 cells, basal expression was detected using the combination ARE-TRE sequence or the ARE, but not the TRE alone, p-Benzoquinone, catechol and hydroquinone, but not resorcinol, induced gene expression in both Hep G2 and F9 cells via the ARE-TRE and ARE sequences, but the TRE sequence did not contribute to this induction. We therefore conclude that induction of human NAD(P)H:quinone oxidoreductase by monofunctional inducers is via the ARE and not the TRE, and that the induction is mediated by proteins other than Fos and Jun.

Amino Acid Sequence↗