Premium
A β‐(1→4)‐xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms
Author(s) -
Urahara Takeshi,
Tsuchiya Kouji,
Kotake Toshihisa,
Tohnooka Takuji,
Komae Kozo,
Kawada Naoyuki,
Tsumuraya Yoichi
Publication year - 2004
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.0031-9317.2004.00390.x
Subject(s) - chemistry , hordeum vulgare , enzyme , biochemistry , microsome , biology , poaceae , botany
A β‐(1→4)‐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 β‐(1→4)‐xylooligosaccharides derivatized at their reducing ends with 2‐aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β‐(1→4)‐xylotriose (Xyl 3 ‐PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl 4−8 ‐PA. Methylation analysis and enzymatic digestions with β‐xylosidase (EC 3.2.1.37) and endo ‐β‐(1→4)‐xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β‐(1→4)‐linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X‐100 and 5 m M MnCl 2 . The apparent Michaelis constant and maximal velocity of the enzyme for Xyl 3 ‐PA were 2.1 m M and 25 400 pmol min −1 mg protein −1 , respectively. The enzyme also transferred [ 14 C]Xyl from UDP‐[ 14 C]Xyl into higher β‐(1→4)‐xylooligosaccharides and birchwood xylans through β‐(1→4)‐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.