Biochemical characterization and molecular cloning of an α-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea
Biochemical characterization and molecular cloning of an α-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea
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DOI：
10.1074/jbc.m000677200
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发表时间：
2000-05-19
影响因子：
4.8
通讯作者：
Keegstra, K
中科院分区：
文献类型：
--
作者：
Faik, A;Bar-Peled, M;Keegstra, K
豌豆微体含有一个α-岩藻糖基转移酶，它将来自GDP-岩藻糖的岩藻糖结合到木葡聚糖中，优先将其添加到离重复亚单位还原端最近的半乳糖残基的2-O位，该酶用洗涤剂增溶，并通过GDP-己醇胺-琼脂糖亲和层析和凝胶过滤纯化。利用从纯化的酶中获得的多肽序列，克隆到一个编码565个氨基酸的蛋白质，预测的相对分子质量为kDa，与拟南芥的同源物有62.3%的同源性。经SDS-聚丙烯酰胺凝胶电泳法测定，纯化后的转移酶迁移率约为63 kDa，但以较高相对分子质量(约250 kDa)的活性蛋白形式从凝胶过滤柱上洗脱出来，表明该酶为低聚物，为低聚木糖所特有，可被低聚木糖和副产物GDP抑制，酶的最适pH为中性，不需要二价离子。动力学分析表明，GDP-岩藻糖和木葡聚糖与酶的结合顺序是随机的。N-乙基马来酰亚胺是一种半胱氨酸专一性修饰剂，虽然其他几种氨基酸修饰剂对活性有强烈的抑制作用，但对活性几乎没有影响。
Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit, This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at similar to 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight (similar to 250 kDa), indicating that the active form is an oligomer, The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP, The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity.