Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13 C NMR spectroscopy, and the degree of esterification was probed by both 13 C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the ␣ and ␥ isoforms when compared with that of the ␤ isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the ␣ and ␥ isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the ␤ isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.Pectins that represent around 30% of the primary plant cell walls play a key role in plant physiology as well as in plant pathology. The general structure of pectic polymers consists of homogalacturonan linear chains (smooth regions) interspersed with highly branched galacturonic chains (hairy regions). Some of the galacturonic residues linked by ␣-1,4 glycosidic bonds are methyl-esterified at the carboxyl group. The degree of methylesterification (DE) 1 varies greatly depending on the plant organ and the degree of differentiation of the cells. Young, plastic cell walls are generally characterized by a high content of highly methylated pectin that decreases in parallel with the loss of extensibility of the walls, whereas the amount of acidic residues increases (1-3). Moreover, the balance between high and low methylated pectins varies inside the wall of a single cell (4) generating microdomains. Not only the number but also the distribution of free, unesterified galacturonate carboxyl groups within the galacturonan regions will control the gellforming capacity of the pectin and thereby the porosity and the extensibility of the apoplasm (5, 6). It is commonly accepted that the polygalacturonic backbone is polymerized in the cisGolgi cisternae, methylesterified in the medial-Golgi, substituted with side chains in the trans-Golgi, and exported to the cell walls as a highly methylated polygalacturonan (7). At a later stage, it is deesterified in muro by cell wall pectinmethylesterases (PMEs). Many proteins exhibiting PME activity have been purified, and their biochemical features such as molecular weight, optimal pH, pI, and substrate specificity have been established (8). In some cases, the corresponding genes have also been cloned and sequenced (9 -11...

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The hydration behavior of two model disaccharides, methyl-alpha-D-maltoside (1) and methyl-alpha-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) cm(2). s(-1); 2: 4.2. 10(-6) cm(2). 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 alpha-(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 alpha-(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 em leader 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.

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SYNOPSIST h e disaccharide arabinobiose (5-O-cu-I.-arabinofuranosyl-tu-I.-arabinofuranose) constitutes the basic repeating structures found in such polysaccharides as arabinan or in the side chains of the hairy regions of pectins. The conformational behavior of aqueous arabinobiose has been investigated by high resolution nmr and computerized molecular modeling. T h e complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics methods using the flexible residue method. In this study, both the puckering of the arabinofuranose rings and the orientations about the glycosidic torsion angles a, q , and ( I : were considered. Some insights into conformational transitions were obtained through molecular dynamics simulation using the CHARMM force field. In parallel, transient nuclear Overhauser effects at 400.13 MHz and long-range vicinal homonuclear and heteronuclear coupling constants have been measured. T h e theoretical nmr data were calculated taking into account all accessible conformations and using averaging methods for both slow and fast internal motions models. T h e data do not support a single conformational model, and only conformational averaging yields the excellent agreement between the observed and simulated parameters. Within the potential energy surfaces computed for the disaccharide, several low energy conformers can be identified. When these conformations are extrapolated to regular polysaccharide structures, they generate chains of arabinan displaying right-and left-handed chirality and a wide range of repeating units per turn of helix. (~8

Thermodynamic and structural properties of two UNCG tetraloops in very short hairpin octamers, 5'-r(GCUUCGGC)-3' and 5'-r(GCUACGGC)-3', have been studied by means of various physical techniques. Melting profiles of both octamers, obtained from UV absorption spectra taken as a function of temperature, are consistent with a monophasic, progressive and completely reversible order-to-disorder transition and confirm their unusual structural stability (Tm > 51 degrees C). The 1H, 13C and 31P NMR chemical shifts and coupling constants of the UACG loop nucleotides are comparable with those reported previously for UUCG loops, i.e. 2'-endo/anti conformation of the second and third nucleotide of the loop as well as the syn orientation of the ultimate guanine base and the A-type double helical conformation of the hairpin stem. Simulation of quantitative NOESY volumes shows that the UACG octamer adopts a very rigid compact structure which is well represented by an average order parameter of 0.9. Three base-pairs and four additional strong hydrogen bonds are undoubtedly responsible for such limited flexibility. Raman and infrared spectra as a function of temperature reflect the order-to-disorder transition, as well. Vibrational conformational markers in low temperature spectra of both octamers indicate the hairpin structure as the major conformer in aqueous phase. These spectra further support the structural features of most of the nucleotides involved in the tetraloops and clearly demonstrate the structural similarities of the phosphodiester backbone in both hairpins. Consequently, on the basis of all present results, one can deduce that the conformational features of the UUCG and UACG tetraloops seem to be inherent to the UNCG type tetraloops, regardless of either the nature of the tetraloop second base or the stem length.