The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.
The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.
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DOI：
10.1074/jbc.m116.743948
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发表时间：
2016-10-14
期刊：
影响因子：
--
通讯作者：
Cuskin F
中科院分区：
文献类型：
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作者：
Labourel A;Crouch LI;Brás JL;Jackson A;Rogowski A;Gray J;Yadav MP;Henrissat B;Fontes CM;Gilbert HJ;Najmudin S;Baslé A;Cuskin F
植物细胞壁的酶促降解是一个重要的生物过程，具有越来越重要的环境和工业意义。木聚糖是植物细胞壁的主要成分，由β-1，4-木糖（Xylp）单元的主链组成，所述单元通常被阿拉伯呋喃糖（Araf）侧链修饰。一个大的五模块酶，CtXyl 5A，先前显示出特异性靶向阿拉伯聚糖。然而，该酶显示的底物识别机制仍不清楚。在这里，我们报告的晶体结构的阿拉伯糖苷酶和酶的配体复合物。数据显示，其中四个蛋白质模块采用刚性结构，这稳定了催化结构域。在晶体结构中不能观察到C-末端非催化碳水化合物结合模块，表明位置灵活性。与Xylp-β-1，4-Xylp-β-1，4-Xylp-[α-1，3-Araf]-β-1，4-Xylp复合的酶的结构表明，将O3连接到活性位点中的木糖的Araf装饰位于紧邻催化中心的口袋（−2* 亚位点）中。−2* 亚位点也可以与Xylp和Arap结合，解释了为什么该酶可以利用木糖和阿拉伯糖作为特异性决定簇。Glu 68、Tyr 92或Asn 139的丙氨酸取代，与阿拉伯糖和木糖侧链在−2* 亚位点相互作用，消除催化活性。在活性位点的远端，木聚糖主链与酶进行有限的非极性接触，并且羟基暴露于溶剂中。这解释了为什么CtXyl 5A能够水解被广泛修饰并且对经典内切木聚糖酶攻击无效的木聚糖。
The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.