Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase

M Saura-Valls, R Faure, S Ragas, K Piens, H Brumer, TT Teeri, S Cottaz, H Driguez, A Planas*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation front a XG donor to a XG or low-molecular-mass XG fragment Lis the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based oil HPCE (high performance capillary electrophoresis), in con, junction with a defined low-molecular-mass XGO {XG oligosaccharicle; (XXXGXXXG, where G = Glc beta 1,4- and X = [Xyl alpha 1,6]Glc beta 1,4-)l as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Popidus tremula x tremuloides (hybrid aspen) was characterized using file donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions. with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism With Substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor Substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.

Original languageEnglish
Pages (from-to)99-106
Number of pages8
JournalBIOCHEMICAL JOURNAL
Volume395
DOIs
Publication statusPublished - 1 Apr 2006
MoE publication typeA1 Journal article-refereed

Keywords

  • assay
  • capillary electrophoresis
  • ping-pong mechanism
  • xyloglucan endotransglycosylase (XET)
  • xyloglucan oligosaccharides (XGOs)
  • MUTANT ENDOGLUCANASE LACKING
  • GLYCOSYL-ENZYME INTERMEDIATE
  • PLANT-CELL WALLS
  • ENDO-TRANSGLYCOSYLASE
  • ENDOXYLOGLUCAN TRANSFERASE
  • CAPILLARY-ELECTROPHORESIS
  • STRUCTURAL-ANALYSIS
  • TROPAEOLUM-MAJUS
  • ARABIDOPSIS
  • SUBSTRATE

Cite this