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 alpha and gamma 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 alpha isoform activity at pH 5.6 and to inhibit the gamma 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 alpha and gamma 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.

Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications In cell wall expansion.

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 13C NMR spectroscopy, and the degree of esterification was probed by both 13C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the alpha and gamma isoforms when compared with that of the beta isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the alpha and gamma 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 beta 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.

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.