Hydrolysis of pectins with di¡erent degrees and patterns of methylation by the endopolygalacturonase of Fusarium moniliforme E. Bonnin a ;  , A. Le Go¡ a , R. Ko « rner b , J. Vigouroux a , P. Roepstor¡ b , J.-F. Thibault a a Unite ¤ de Recherche sur les Polysaccharides, leurs Organisations et Interactions, INRA, B.P. 71627, 44316 Nantes Cedex 03, France b Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark Received 3 July 2001; received in revised form 10 December 2001; accepted 13 December 2001 Abstract The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : Endopolygalacturonase ; Blockwise methylated pectin ; Randomly methylated pectin ; Homogalacturonan ; Fusarium moniliforme 1. Introduction Pectins are one of the major components in pri- mary cell walls. They are mainly composed of two subunits, namely homogalacturonans (‘smooth’ re- gions) and rhamnogalacturonans (‘hairy’ regions). In homogalacturonan (HG), D-galacturonic acid (GalA) is K 1^4 linked and may be methyl-esteri¢ed on the carboxylic group. The degree of methylation (DM) is de¢ned as the percentage of carboxyl groups esteri¢ed with methanol. In native pectins, DM can be often up to 70^80. On the other hand, O-2 and O-3 of GalA can be acetylated, and the degree of acetylation (DAc) is de¢ned as the percentage of GalA residues esteri¢ed with one acetyl group. Rhamnogalacturonans are mainly constituted of a repeating sequence of [-2) K-Rha (1^4) K-GalA (1] in which L-rhamnose carries some neutral side chains of arabinose and/or galactose. Production of pectin-degrading enzymes by fun- gal pathogens is considered as an important re- quirement for colonisation of plant tissue, since the cleavage of pectins could be a prerequisite for 0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4838(02)00207-8 Abbreviations : DAc, degree of acetylation ; DM, degree of methylation ; DP, degree of polymerisation ; GalA, galacturonic acid; Hg, homogalacturonan ; PG, polygalacturonase ; PME, pectin methyl-esterase * Corresponding author. Fax : +33-2-4067-5066. E-mail address : bonnin@nantes.inra.fr (E. Bonnin). Biochimica et Biophysica Acta 1596 (2002) 83^94 www.bba-direct.com