Catherine Hervé du Penhoat

Isolation and an N.M.R. study of pectins from flax (linum usitatissimum L.)

Abstract The EDTA-extracted pectins from flax, which were fractionated by exclusion chromatography according to size, contained galactose, galacturonic acid, rhamnose, glucose, and traces of arabinose. The main components of the major fraction, a high-molecular-weight galactan, were →4)-β-Galp-(1→ chains and β-Galp non-reducing terminal units according to 1D 1H- and 13C-n.m.r. analysis. The low-molecular-weight fractions contained notable amounts of rhamnose and galacturonic acid. On the basis of multipulse n.m.r. (CHORTLE, COSY, and NOESY), a rhamno-galacturonan structure of type I with β-Galp units attached to the HO-4 of rhamnose can be proposed. This polymer probably constitutes the backbone of the high-molecular-weight polysaccharides.

Ellagitannins in woods of sessile oak and sweet chestnut dimerization and hydrolysis during wood ageing

Abstract Monomeric and dimeric C -glucosidic ellagitannins and phenolic acids were studied by reverse-phase HPLC and gel permeation chromatography in various sapwood and heartwood samples of sessile oak ( Quercus petraea ) and sweet chestnut ( Castanea sativa ). Sessile oak differs from sweet chestnut by the presence in heartwood of pentose-substituted ellagitannins. Analysis of ellagitannin concentrations and a model experiment show that vescalagin is a common precursor of all dimers which are likely formed by a non-enzymatic reaction. Ellagic acid and gallic acid in oak and chestnut woods result, respectively, from the hydrolysis of ellagitannins and some unknown galloyl esters. Both dimerization and hydrolysis occur at the sapwood-heartwood transition and in the dead heartwood.

New investigations of the structure of grape arabino-galactan-protein

The structure of an arabinogalactan-protein (AGP) isolated from grape juice was studied by methylation analysis, n.m.r. spectroscopy, and interactions with peanut lectin, after specific degradation with purified enzymes and/or Smith degradation. AGP appeared to be homogeneous with a weight-average molecular weight of 110,000. Treatment of AGP with arabinofuranosidase released 88% of the arabinose and left GP1. Hydrolysis of GP1 with an endo-(1----6)-beta-D-galactanase removed 50% of the galactose and left GP2. Smith degradation of GP1 gave a 3-linked galactan that still contained 3,6-linked residues. Endogalactanase- and Smith-degraded GP1, but not AGP and GP1, reacted strongly with peanut lectin. Thus, AGP is a 3-linked galactan cross-linked at positions 6. The core also carries, at positions 6, 6-linked galactan chains heavily 3-substituted with arabinofuranose residues.

Structural elucidation of new dimeric ellagitannins from Quercus robur L. roburins A–E

Eight ellagitannins from Quercus robur L. wood have been studied by high-resolution 1H and 13C NMR and FAB-MS spectroscopy. Three of these polyphenols are known compounds, namely, castalagin, vescalagin, and grandinin. Five of the ellagitannins are new oligomeric compounds containing vescalagin or castalagin moieties bonded to a pentose, lyxose or xylose. The inter-unit linkages are carbon–carbon bonds between C-1 of the glucosyl residue of one unit and either the C-2′ of the hexahydroxydiphenoyl (HHDP) group of a second unit or the C-1 of a pentosyl sugar.

A hydration study of (1→4) and (1→6) linked α‐glucans by comparative 10 ns molecular dynamics simulations and 500‐MHz NMR

The hydration behavior of two model disaccharides, methyl‐α‐D‐maltoside (1) and methyl‐α‐D‐isomaltoside (2), has been investigated by a comparative 10 ns molecular dynamics study. The detailed hydration of the two disaccharides was described using three force fields especially developed for modeling of carbohydrates in explicit solvent. To validate the theoretical results the two compounds were synthesized and subjected to 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements (1: 4.0 · 10−6 cm2 · s−1; 2: 4.2 · 10−6 cm2 · s−1). In short, the older CHARMM‐based force field exhibited a more structured carbohydrate–water interaction leading to better agreement with the diffusional properties of the two compounds, whereas especially the α‐(1→6) linkage and the primary hydroxyl groups were inaccurately modeled. In contrast, the new generation of the CHARMM‐based force field (CSFF) and the most recent version of the AMBER‐based force field (GLYCAM‐2000a) exhibited less structured carbohydrate–water interactions with the result that the diffusional properties of the two disaccharides were underestimated, whereas the simulations of the α‐(1→6) linkage and the primary hydroxyl groups were significantly improved and in excellent agreement with homo‐ and heteronuclear coupling constants. The difference between the two classes of force field (more structured and less structured carbohydrate–water interaction) was underlined by calculation of the isotropic hydration as calculated by radial pair distributions. At one extreme, the radial O…O pair distribution function yielded a peak density of 2.3 times the bulk density in the first hydration shell when using the older CHARMM force field, whereas the maximum density observed in the GLYCAM force field was calculated to be 1.0, at the other extreme.

The three-dimensional structure of the mega-oligosaccharide rhamnogalacturonan II monomer: a combined molecular modeling and NMR investigation.

In this study, we describe the first optimized molecular models of the mega-oligosaccharide rhamnogalaturonan II, that is found in the primary cell walls of all higher plants. The 750 MHz 1H NMR data previously reported and new heteronuclear correlation spectra (sensitivity-enhanced HSQC and HSQC-TOCSY) were first reassigned in light of the modifications in the primary structure. In turn, the experimental NMR data revealed the presence of an additional sugar, alpha-Araf (E-chain), and also the disaccharidic repeating unit of RG-I, another component of the pectic matrix. Due to a fuller picture of the primary structure of RG-II, a much more complete assignment of the NOE data has been achieved. A systematic computational study based on these NOEs lead us to a realistic three-dimensional description of the RG-II, in excellent agreement with the molecular dimensions obtained from various experimental methods.

Travelling on the potential energy surfaces of carbohydrates: comparative application of an exhaustive systematic conformational search with an heuristic search

The calculated ensembles found by a heuristic conformational search algorithm, CICADA, for three small carbohydrates, ethyl beta-lactoside, methyl alpha-D-galactoside, and methyl beta-D-galactoside, are evaluated in terms of their ability to reproduce time-averaged optical rotation and NMR data. A unique dynamic model for methyl beta-D-galactoside has been obtained by fitting experimental NOESY volumes to the theoretical ones elaborated from the CICADA ensemble internuclear distances with the model-free formalism. In the case of ethyl beta-lactoside, the CICADA ensemble is compared to that of an exhaustive systematic grid-search method. The CICADA algorithm proved to be a very efficient method to find most of the important minima on even very complex potential energy surfaces, and the spectral quality of the CICADA ensemble was found to be of equal quality, if not superior, to that of the exhaustive systematic grid-search method. The CICADA algorithm has several advantages over other conformational search algorithms: (1) It has polynomial dependence of dimensions on computer time in contrast to the grid search, which has exponential dependence, (2) the conformations found are free of artificial harmonic constraint potentials, (3) it passes all barriers amongst families of conformations on conformational hypersurface but spends almost all its time in the essential highly populated areas, (4) the inherent properties of the algorithm make rigorous minimization criteria superfluous and provide good convergence behavior, and (5) as an important spin-off, it provides low-energy interconversion pathways that can, amongst others, be used for estimating adiabatic rotational barriers.

Modelling of arabinofuranose and arabinan. Part 1: conformational flexibility of the arabinofuranose ring

Abstract The conformational behaviour of methyl β- d - and methyl α- l -arabinofuranosides have been assessed through computations performed with the molecular mechanics program MM3 using the flexible residue method. Energies for various envelope and twist conformers were calculated as a function of the puckering parameters Q and Φ. The gauche-gauche, gauche-trans, and trans-gauche orientations of the primary hydroxyl groups at C-5 were accounted for. Our calculations provide some insight into extensive conformational flexibility that both molecules display and shed light on the possible pathways for conformational interconversions. Both molecules display very characteristic conformational behaviour. The conformations of methyl β- d -arabinofuranoside, which have been determined by single-crystal X-ray diffraction studies, are distributed in two fairly deep low-energy wells. These conformations are no more than 1.5 kcal/mol above the respective energy minima. In the case of methyl α- l -arabinofuranoside, crystallographic data are lacking and only 1 H- 1 H coupling constants are available. Using suitable equations for the H-C-C-H segments, the theoretical 3 J H-H coupling constants were calculated as a function of the two puckering parameters, taking into account all the accessible conformations. Agreement with the experimentally reported data confirms the high flexibility of furanose rings in solution.

Development of arabinans and galactans during the maturation of hypocotyl cells of mung bean (Vigna radiata Wilczek)

Abstract Hypocotyl cell walls contain galactans and arabinans that are soluble in boiling water. During maturation, the Ara/Gal ratio remains unchanged but high-molecular-weight galactans are replaced by smaller polymers. On the basis of the 1 H-n.m.r. 2D-COSY(δ-δ, 1 H- 1 H)n.m.r., and 13 C-n.m.r. spectra, a (1→5)-α-Ara f structure can be proposed for the arabinans in both young and nature cell walls. However, the galanctan(s) changed from a probably highly branched to an unbranched (1→4)-β-Gal p structure during maturation.

Control of Mung Bean Pectinmethylesterase Isoform Activities INFLUENCE OF pH AND CARBOXYL GROUP DISTRIBUTION ALONG THE PECTIC CHAINS

Well-characterized pectin samples with a wide range of degrees of esterification (39–74%) were incubated with the solubilized pure α and γ isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the α isoform activity at pH 5.6 and to inhibit the γ isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 ± 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem.273, 33150–33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of α and γ isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed.