Dataset Information

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

ABSTRACT: Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as 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 on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} 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 Populus tremula x tremuloides (hybrid aspen) was characterized using the 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.

SUBMITTER: Saura-Valls M

PROVIDER: S-EPMC1409682 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Saura-Valls Marc M Fauré Régis R Ragàs Sergi S Piens Kathleen K Brumer Harry H Teeri Tuula T TT Cottaz Sylvain S Driguez Hugues H Planas Antoni A

The Biochemical journal 20060401 1

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as 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 poly ...[more]

PMID: 16356166

Similar Datasets

Project description:1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that it thus causes the wall-loosening required for plant cell expansion.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets