A xylosyltransferase that synthesizes beta-(1 -> 4)-xylans in wheat (Triticum aestivum L.) seedlings

A particulate preparation from 6-day-old seedlings of wheat (Triticum aesti vum L.) was found to contain a xylosyltransferase (XylTase) which incorpora ted xylose (Xyl) from UDP-xylose into exogenous beta-(1-->4)-xylooligosacch arides with 2-aminopyridine-derivatized reducing end groups. High-performan ce liquid chromatographic analysis showed that the chain elongation of pyri dylaminated beta-(1-->4)-xylotriose (XYl(3)-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate conc entrations and reaction times. Methylation analysis and beta -xylosidase di gestion of the newly synthesized XYl(4)-PA confirmed that the xylosyl resid ues were incorporated through beta-(1-->4)-linkages. The enzyme was maximal ly active at pH 6.8 and 20 degreesC, and required Triton X-100, which enhan ced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with inc reasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl(5)-PA was almost 7 times as efficient as Xyl(2)-PA. The appar ent Michaelis constants of the enzyme for Xyl(3)-PA and UDP-xylose were 13. 5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radi oactive sugars (Xyl together with a small portion Of L-arabinose) from UDP[ C-14]xylose into higher beta-(1-->4)-xylooligosaccharides (degree of polyme rization > 7) with or without (4-O-methyl-)glucuronosyl side chains at acti vities comparable to those observed for pyridylaminated xylooligosaccharide s, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta -xylanase degraded it into mainly xyl obiose, providing further evidence that the xylosyl residues are incorporat ed through beta-(1-->4)-linkages.