Vasorelaxant activity of twenty-one physiologically relevant (poly)phenolic metabolites on isolated mouse arteries.

Polyphenols are beneficial for health, but are metabolised after consumption. We compared the vasorelaxant capacity of twenty-one physiologically relevant polyphenol metabolites in isolated mouse arteries. Hesperetin, urolithins and ferulic acid-4-O-sulfate - not their glucuronidated forms or ferulic acid - caused vasorelaxation. Therefore, we advise the use of relevant conjugates in future mechanistic research.

The pharmacokinetics of anthocyanins and their metabolites in humans.

BACKGROUND AND PURPOSE: Anthocyanins are phytochemicals with reported vasoactive bioactivity. However, given their instability at neutral pH, they are presumed to undergo significant degradation and subsequent biotransformation. The aim of the present study was to establish the pharmacokinetics of the metabolites of cyanidin-3-glucoside (C3G), a widely consumed dietary phytochemical with potential cardioprotective properties. EXPERIMENTAL APPROACH: A 500 mg oral bolus dose of 6,8,10,3',5'-(13)C5-C3G was fed to eight healthy male participants, followed by a 48 h collection (0, 0.5, 1, 2, 4, 6, 24, 48 h) of blood, urine and faecal samples. Samples were analysed by HPLC-ESI-MS/MS with elimination kinetics established using non-compartmental pharmacokinetic modelling. KEY RESULTS: Seventeen (13)C-labelled compounds were identified in the serum, including (13)C5-C3G, its degradation products, protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), 13 metabolites of PCA and 1 metabolite derived from PGA. The maximal concentrations of the phenolic metabolites (Cmax ) ranged from 10 to 2000 nM, between 2 and 30 h (tmax) post-consumption, with half-lives of elimination observed between 0.5 and 96 h. The major phenolic metabolites identified were hippuric acid and ferulic acid, which peaked in the serum at approximately 16 and 8 h respectively. CONCLUSIONS AND IMPLICATIONS: Anthocyanins are metabolized to a structurally diverse range of metabolites that exhibit dynamic kinetic profiles. Understanding the elimination kinetics of these metabolites is key to the design of future studies examining their utility in dietary interventions or as therapeutics for disease risk reduction.

Hydroxytyrosyl ethyl ether exhibits stronger intestinal anticarcinogenic potency and effects on transcript profiles compared to hydroxytyrosol.

The anticarcinogenic activity of hydroxytyrosyl ethyl ether (HTy-Et) compared to its precursor hydroxytyrosol (HTy) has been studied in human Caco-2 colon adenocarcinoma cells. 451 and 977 genes were differentially expressed in Caco-2 cells exposed to HTy or HTy-Et for 24h, respectively, compared with untreated cells (P<0.005; FDR=0), using Affymetrix microarrays. Results showed that both HTy and HTy-Et inhibited cell proliferation and arrested the cell cycle by up-regulating p21 and CCNG2 and down-regulating CCNB1 protein expression. HTy and HTy-Et also altered the transcription of specific genes involved in apoptosis, as suggested by the up-regulation of BNIP3, BNIP3L, PDCD4 and ATF3 and the activation of caspase-3. Moreover, these polyphenols up-regulated xenobiotic metabolizing enzymes UGT1A10 and CYP1A1, enhancing carcinogen detoxification. In conclusion, these results highlight that HTy and its derivative HTy-Et modulate molecular mechanisms involved in colon cancer, with HTy-Et being more effective than HTy.

Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)-epicatechin are poor substrates for commercial aryl-sulfatases: implications for studies concerned with quantifying epicatechin bioavailability.

Epicatechin is a widely consumed dietary flavonoid and there is substantial evidence that it contributes to the health benefits reported for flavanol-rich cocoa products including dark chocolate. Numerous reports have described the appearance of epicatechin and epicatechin phase-2 conjugates (sulfates and glucuronides of epicatechin and methylepicatechin) in blood and urine samples of subjects following ingestion of epicatechin. The most widely reported method of quantifying total epicatechin in plasma and urine samples involves hydrolysis with a mixture of ß-glucuronidase and sulfatase to convert the conjugates to epicatechin aglycone which is subsequently quantified. We observed a lack of hydrolysis of epicatechin sulfates and methylepicatechin sulfates using commercial sulfatases and investigated this further. Samples of urine or plasma from subjects who had consumed epicatechin were subjected to enzyme hydrolysis and then analysed using LC-MS/MS, or analysed without enzyme hydrolysis. Attempts to increase the extent of hydrolysis of epicatechin conjugates were made by increasing the amount of enzyme, hydrolysis pH and length of incubations, and using alternative sources of enzyme. The standard hydrolysis conditions failed to hydrolyse the majority of epicatechin sulfates and methylepicatechin sulfates. Even when the quantity of enzyme and incubation period was increased, the pH optimised, or alternative sources of sulfatases were used, epicatechin monosulfates and methylepicatechin monosulfates remained as major peaks in the chromatograms of the samples. An assessment of literature data strongly suggested that the majority of reports where enzyme hydrolysis was used had significantly underestimated epicatechin bioavailability in humans. Methods for quantifying epicatechin concentrations in blood and urine need to take account of the lack of hydrolysis of (methyl)epicatechin-sulfates, for example by quantifying these directly using LC-MS/MS.

EuroFIR eBASIS: application for health claims submissions and evaluations.

BACKGROUND: The European Food Information Resource (EuroFIR) network has established the eBASIS (Bioactive Substances in Food Information System) online food composition and biological effects database for plant-derived bioactive compounds (phytochemicals). On the basis of submitted evidence, the European Food Safety Authority (EFSA) expert panel on Dietetic Products, Nutrition and Allergies assesses whether claims made under articles 13.1, 13.5 or 14 of the Regulation (EC) 1924/2006, which governs the use of nutrition and health claims on foods, are scientifically justified. This report evaluates the eBASIS biological effects database in the preparation and evaluation of health claims dossiers. METHODS: The eBASIS biological effects database is a compilation of expert-evaluated data extracted from the literature, prioritizing human intervention studies to investigate health effects of phytochemicals. Currently included are >750 records from 445 studies providing data on 56 validated biomarkers, mainly relating to cardio-metabolic and bone health outcomes. The data cover 144 bioactive compounds from 17 compound classes. Using the EFSA Register of Questions and the database of general function health claims, we identified claims relating to phytochemicals made under articles 13.1, 13.5 and 14 and compared them with the eBASIS database to identify overlap between them. RESULTS: The EFSA online health claims database contains 4240 submissions under article 13.1, of which 2157 pertain to plants or plant-based bioactive compounds; 496 of these relate to plants or bioactive compounds included in the eBASIS biological effects database. Out of the 18 current 13.5 'new function' claims on EFSA's register of questions, 7 are for plants or plant-based bioactive compounds, of which 6 are included in eBASIS. Of the 222 defined article 14 claims, 21 pertain to plants or plant-based bioactive compounds, of which 19 are in eBASIS. CONCLUSIONS: There is extensive overlap between eBASIS and the submitted health claims that relate to plant-based bioactive compounds. EuroFIR eBASIS is a useful tool for regulators to independently check completeness of health claims applications relating to phytochemicals and is a potentially valuable resource to assist claimants in the compilation of dossiers on functional foods and health claims.

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery.

BACKGROUND AND PURPOSE: Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery. EXPERIMENTAL APPROACH: Segments of the porcine coronary artery were prepared for either isometric tension recording or determination of cyclic GMP content. The effect of quercetin and metabolites on submaximal responses to U46619 was examined in the presence and absence of substance P, bradykinin, forskolin, sodium nitroprusside (SNP) and glyceryl trinitrate (GTN). KEY RESULTS: Quercetin and quercetin 3'-sulphate inhibited endothelin and U46619-induced contractions with greater potency (three- to fivefold) against the former, while quercetin 3-glucoronide was inactive. Quercetin enhanced both the cyclic GMP content of the artery (threefold) and cyclic GMP-dependent relaxations to GTN and SNP (two to threefold), but forskolin-induced relaxations were unaffected. Although the effect of quercetin was qualitatively similar to that noted for UK-114,542, a selective inhibitor of phosphodiesterase 5, it was still evident against SNP-induced relaxations in the presence of 10 nM UK-114,542. Quercetin and quercetin 3'-sulphate significantly reduced the development of GTN-associated 'tolerance'. CONCLUSIONS AND IMPLICATIONS: Quercetin and quercetin 3'-sulphate inhibited receptor-mediated contractions of the porcine isolated coronary artery by an endothelium-independent action. Quercetin selectively enhanced cyclic-GMP-dependent relaxations by a mechanism not involving phosphodiesterase 5 inhibition. In addition, quercetin and quercetin 3'-sulphate opposed GTN-induced tolerance in vitro, which may be beneficial for patients treated for angina pectoris.

The procyanidin-mediated induction of apoptosis and cell-cycle arrest in esophageal adenocarcinoma cells is not dependent on p21(Cip1/WAF1).

Previous studies have shown that the proanthocyanidin-mediated induction of apoptosis and arrest of the cell cycle in cancer cells was associated with up-regulation of p21(Cip1/WAF1) (p21), suggesting that p21 may be the molecular mediator of the observed effects. Here we show that procyanidins induce a rapid and sustained arrest of the cell cycle, and increase apoptosis, concomitant with an increase in p21 expression. However, blocking the PA-induced up-regulation of p21 expression with siRNA did not alter PA-mediated changes in apoptosis and cell cycle, demonstrating that p21 is not responsible for the PA-induced effects.

Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans.

OBJECTIVE: To determine the absorption, excretion and metabolism of kaempferol in humans. DESIGN: A pharmacokinetic study of kaempferol from endive over 24 h. SUBJECTS: Four healthy males and four healthy females. RESULTS: Kaempferol, from a relatively low dose (9 mg), was absorbed from endive with a mean maximum plasma concentration of 0.1 microM, at a time of 5.8 h, indicating absorption from the distal section of the small intestine and/or the colon. Although a 7.5-fold interindividual variation between the highest and lowest maximum plasma concentration was observed, most individuals showed remarkably consistent pharmacokinetic profiles. This contrasts with profiles for other flavonoids that are absorbed predominantly from the large intestine (eg rutin). An average of 1.9% of the kaempferol dose was excreted in 24 h. Most subjects also showed an early absorption peak, probably corresponding to kaempferol-3-glucoside, present at a level of 14% in the endive. Kaempferol-3-glucuronide was the major compound detected in plasma and urine. Quercetin was not detected in plasma or urine indicating a lack of phase I hydroxylation of kaempferol. CONCLUSIONS: Kaempferol is absorbed more efficiently than quercetin in humans even at low oral doses. The predominant form in plasma is a 3-glucuronide conjugate, and interindividual variation in absorption and excretion is low, suggesting that urinary kaempferol could be used as a biomarker for exposure.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum.

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the beta-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals.

Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye and wheat brans. Hydrolysis by intestinal esterase(s) is very likely the major route for release of antioxidant hydroxycinnamic acids in vivo.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor.

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear 1H-15N NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta-strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. 15N longitudinal and transverse relaxation rates, and [1H]-15N heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85+/-0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides.

Intestinal release and uptake of phenolic antioxidant diferulic acids.

Diferulic acids are potent antioxidants and are abundant structural components of plant cell walls, especially in cereal brans. As such, they are part of many human and animal diets and may contribute to the beneficial effect of cereal brans on health. However, these phenolics are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. This study provides the first evidence that diferulic acids can be absorbed via the gastrointestinal tract. The 5-5-, 8-O-4-, and 8-5-diferulic acids were identified in the plasma of rats after oral dosing with a mixture of the three acids in oil. Our study also reveals that human and rat colonic microflora contain esterase activity able to release 5-5-, 8-O-4-, and 8-5-diferulic acids from model compounds and dietary cereal brans, hence providing a mechanism for release of dietary diferulates prior to absorption of the free acids. In addition, cell-free extracts from human and rat small intestine mucosa exhibited esterase activity towards diferulate esters. Hence, we have shown that esterified diferulates can be released from cereal brans by intestinal enzymes, and that free diferulic acids can be absorbed and enter the circulatory system. Our results suggest that the phenolic antioxidant diferulic acids are bioavailable.

A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module.

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.

Three-dimensional NMR structure of the sixth ligand-binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities.

The sixth ligand-binding module of the low-density lipoprotein receptor contributes to the binding of apolipoprotein B100-containing lipoproteins. 1H NMR spectroscopy, DYANA and X-PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E-containing lipoproteins in addition to apolipoprotein B100-containing lipoproteins.

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Purification of cytosolic beta-glucosidase from pig liver and its reactivity towards flavonoid glycosides.

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic beta-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic beta-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4'-glucoside was a substrate, but neither quercetin 3, 4'-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K(m) ranged from 25 to 90 microM while those for V(max) were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4'-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic beta-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.

A modular cinnamoyl ester hydrolase from the anaerobic fungus Piromyces equi acts synergistically with xylanase and is part of a multiprotein cellulose-binding cellulase-hemicellulase complex.

A collection of clones, isolated from a Piromyces equi cDNA expression library by immunoscreening with antibodies raised against affinity purified multienzyme fungal cellulase-hemicellulase complex, included one which expressed cinnamoyl ester hydrolase activity. The P. equi cinnamoyl ester hydrolase gene (estA) comprised an open reading frame of 1608 nt encoding a protein (EstA) of 536 amino acids and 55540 Da. EstA was modular in structure and comprised three distinct domains. The N-terminal domain was closely similar to a highly conserved non-catalytic 40-residue docking domain which is prevalent in cellulases and hemicellulases from three species of anaerobic fungi and binds to a putative scaffolding protein during assembly of the fungal cellulase complex. The second domain was also not required for esterase activity and appeared to be an atypically large linker comprising multiple tandem repeats of a 13-residue motif. The C-terminal 270 residues of EstA contained an esterase catalytic domain that exhibited overall homology with a small family of esterases, including acetylxylan esterase D (XYLD) from Pseudomonas fluorescens subsp. cellulosa and acetylxylan esterase from Aspergillus niger. This region also contained several smaller blocks of residues that displayed homology with domains tentatively identified as containing the essential catalytic residues of a larger group of serine hydrolases. A truncated variant of EstA, comprising the catalytic domain alone (EstA'), was expressed in Escherichia coli as a thioredoxin fusion protein and was purified to homogeneity. EstA' was active against synthetic and plant cell-wall-derived substrates, showed a marked preference for cleaving 1-->5 ester linkages between ferulic acid and arabinose in feruloylated arabino-xylo-oligosaccharides and was inhibited by the serine-specific protease inhibitor aminoethylbenzene-sulphonylfluoride. EstA' acted synergistically with xylanase to release more than 60% of the esterified ferulic acid from the arabinoxylan component of plant cell walls. Western analysis confirmed that EstA is produced by P. equi and is a component of the aggregated multienzyme cellulase-hemicellulase complex. Hybrid proteins, harbouring one, two or three iterations of the conserved 40-residue fungal docking domain fused to the reporter protein glutathione S-transferase, were produced. Western blot analysis of immobilized P. equi cellulase-hemicellulase complex demonstrated that each of the hybrid proteins bound to a 97 kDa polypeptide in the extracellular complex.

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.