A b-(14)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A b-(14)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley (Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous b-(14)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated b-(14)-xylotriose (Xyl(3)(4-8)(2)(3)(-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl)(-PA. Methylation analysis and enzymatic digestions with b-xylosidase (EC 3.2.1.37) and endo-b-(14)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through b-(14)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20C and most enhanced in the presence of 0.5% Triton X-100 and 5 mM MnCl)(. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl)-PA were 2.1 mM and 25 400 pmol min-1 mg protein-1, respectively. The enzyme also transferred [14C]Xyl from UDP-[14C]Xyl into higher b-(14)-xylooligosaccharides and birchwood xylans through b-(14)-linkages. The enzyme activity varied according to the stage of development (7-35 days after flowering) of the endosperms. Maximal activity occurred at 13-16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11-22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.