The first glycosynthase derived from an inverting glycoside hydrolase
The first glycosynthase derived from an inverting glycoside hydrolase
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DOI：
10.1074/jbc.m511202200
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发表时间：
2006-01-20
影响因子：
4.8
通讯作者：
Kitaoka, M
中科院分区：
文献类型：
--
作者：
Honda, Y;Kitaoka, M
还原端木糖释放外切低聚木聚糖酶（雷克斯，EC 3.2.1.156）是一种转化GH，其水解低聚木糖（>= X-3）以在其还原端释放X-1。野生型酶表现出Hehre再合成水解机制，其中在X-1作为受体分子的存在下，α-X2 F水解为X-2和HF。然而，在反应中未检测到转糖苷化产物（X-3）。为了将还原末端木糖释放外切寡聚木聚糖酶转化为糖合酶，通过饱和随机诱变构建了催化碱基（Asp-263）突变的衍生物。发现9个氨基酸残基突变体（Asp-263至Gly、Ala、瓦尔、Thr、Leu、Asn、Cys、Pro或Ser）具有从α-X2 F和X-1形成X-3的糖合酶活性。其中，D263 C显示出最高水平的X-3产量，而D263 N显示出最快的α-X2 F消耗。D263 C突变体显示出比D263 N低10倍的水解活性，导致X-3的最高产量。X-2是由D263 C突变体反应的早期阶段形成的，这表明通过缩合形成的X-3的一部分在其从酶释放之前被水解。为了从转化酶获得糖合酶活性，重要的是使F-释放活性的降低最小化，同时使水解活性的降低最大化。本研究扩大了从转化酶转化糖苷酶的可能性。
Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (>= X-3) to release X-1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X-2 and HF in the presence of X-1 as an acceptor molecule. However, the transglycosidation product (X-3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X-3 from alpha-X2F and X-1. Among them, D263C showed the highest level of X-3 production, and D263N exhibited the fastest consumption of alpha-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X-3. X-2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X-3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F--releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.