New insights into the role of the thumb-like loop in GH-11 xylanases
New insights into the role of the thumb-like loop in GH-11 xylanases
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DOI：
10.1093/protein/gzl049
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发表时间：
2007-01-01
影响因子：
2.4
通讯作者：
O'Donohue, Michael J.
中科院分区：
文献类型：
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作者：
Paes, Gabriel;Tran, Vinh;O'Donohue, Michael J.
GH-11木聚糖酶具有高度特异性，并具有拇指形环，这是具有卷曲支架的酶中的独特结构。为了研究这种结构，对来自Thermobacillus xylanilyticus的GH-11木聚糖酶（Tx-Xyl）进行体外诱变。靶标是位于拇指尖端的保守氨基酸Pro（114）-Ser（115）-Ile（116）以及Thr（121）和Tyr（111），连接拇指与主要酶支架的接头残基。位点饱和诱变提供了一种具有新三联体（Pro（114）-Gly（115）-Cys（116））的活性变体，该三联体在天然存在的GH-11木聚糖酶中未发现。该突变体催化木聚糖水解的k（cat）值提高了20%。通过删除接头残基重新定位拇指产生不同的效果。如通过计算机模拟分析预测的，Thr（121）的缺失对活性具有严重后果，而Tyr（111）的缺失仅影响（4倍降低）k（cat）。最后，使用缺失诱变来产生几乎无催化活性的无拇指变体。木四糖和木五糖的荧光滴定显示，这种拇指缺失的木聚糖酶保留了结合底物的能力。这种结合与野生型酶的结合相当。此外，与野生型Tx-Xyl不同，拇指缺失的木聚糖酶有效地结合纤维四糖，尽管没有检测到纤维素水解活性。总的来说，这些数据表明拇指是底物选择的关键决定因素，并支持先前的数据，表明它在催化过程中发挥作用。
GH-11 xylanases are highly specific and possess a thumb-shaped loop, a unique structure among enzymes with a jelly-roll scaffold. To investigate this structure, in vitro mutagenesis was performed on a GH-11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus. Targets were the conserved amino acids Pro(114)-Ser(115)-Ile(116) that are located at the thumb's tip and Thr(121) and Tyr(111), linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro(114)-Gly(115)-Cys(116)), not found in naturally occurring GH-11 xylanases. The k(cat) value for xylan hydrolysis catalysed by this mutant was increased by 20%. Re-positioning of the thumb through the deletion of the linker residues produced different effects. As predicted by in silico analyses, deletion of Thr(121) had drastic consequences on activity, whereas deletion of Tyr(111) only affected (4-fold decrease) k(cat). Finally, deletion mutagenesis was used to create a thumbless variant that was almost catalytically inactive. Fluorescence titration with xylotetraose and xylopentaose revealed that this thumb-deleted xylanase retained the ability to bind substrates. This binding was comparable to that of the wild-type enzyme. Additionally, unlike wild-type Tx-Xyl, the thumb-deleted xylanase efficiently bound cellotetraose, although no cellulose hydrolysing activity was detected. Overall, these data show that the thumb is a key determinant for substrate selection and support previous data that suggest that it plays a role in the catalytic process.