Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase
Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase
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DOI：
10.1093/glycob/cwn011
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发表时间：
2008-04-01
期刊：
影响因子：
4.3
通讯作者：
Kitaoka, Motomitsu
中科院分区：
文献类型：
--
作者：
Honda, Yuji;Fushinobu, Shinya;Kitaoka, Motomitsu
已知酪氨酸残基Y198在还原末端释放木糖的外寡聚糖酶(REX)中支持具有一般碱基残基D263的亲核水分子。酪氨酸残基的突变将其转变为苯丙氨酸，导致在木糖存在下，α-木糖基氟化物的水解性急剧下降，释放氟的活性略有增加。相反，D263位突变导致F-释放活性降低。由于Y198F具有较高的释氟活性和较低的水解性，在糖合酶反应过程中积累了大量的产物。我们提出了一种新的方法来从转化糖苷水解酶中产生糖合成酶，方法是在保持普通碱基不变的情况下，将含有亲核水分子的残基与普通碱基一起突变。
The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F-releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F-releasing activity. As a result of the high F-releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.