To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) along the donor substrate chain, we incubated the enzymes with tamarind (Tamarindus indica) xyloglucan (donor substrate; ≈ 205kDa; 21µM) plus the nonasaccharide [3H]XLLGol (Gal2·Xyl3·Glc3· [3H]glucitol; acceptor substrate; 0.6µM). After short incubation times, to minimize multiple cleavages, the size of the 3H-labelled transglycosylation products (determined by gel-permeation chromatography) indicated the positions of the cleavage sites relative to the non-reducing terminus of the donor. There was very little difference between the size profiles of the products formed by any of ten XETs tested [one native XET purified from cauliflower (Brassica oleracea) florets, four native XET isoenzymes purified from etiolated mung-bean (Phaseolus aureus) shoots, native XETs purified from lentil (Lens culinaris) and nasturtium (Tropaeolum majus) seeds, and three insect-cell-produced thale-cress (Arabidopsis thaliana) XETs (EXGT, TCH4 and MERI-5)]. All such product profiles showed a good fit to a model in which the enzyme chooses its donor substrate independently of size and attacks it, once only, at a randomly selected cleavage site. The results therefore do not support the hypothesis that different XET isoenzymes are adapted to produce longer or shorter products such as might favour either the efficient integration of new xyloglucan into the cell wall or the re-structuring of old xyloglucan within an expanding wall.

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