Evidence that GH115 α-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility.
Evidence that GH115 α-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility.
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DOI：
10.1074/jbc.m113.525295
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发表时间：
2014-01-03
期刊：
影响因子：
--
通讯作者：
Bolam DN
中科院分区：
文献类型：
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作者：
Rogowski A;Baslé A;Farinas CS;Solovyova A;Mortimer JC;Dupree P;Gilbert HJ;Bolam DN
背景：GH 115葡萄糖醛酸苷酶从木聚糖链上去除葡萄糖醛酸的结构是未知的。结果：卵形拟杆菌GH 115葡萄糖醛酸酶是一种二聚体酶，含有一个灵活的活性位点口袋。结论：葡萄糖醛酸酶催化器的组装需要大量的构象变化。意义：糖苷水解酶的构象变化是非常罕见的。植物细胞壁的微生物降解是一个重要的生物过程，与环境重要的工业如生物能源和生物精炼部门高度相关。壁的主要成分是葡糖醛酸木聚糖，一种β 1，4-连接的木糖多糖，用α-连接的葡糖醛酸和/或甲基葡糖醛酸（GlcA/MeGlcA）修饰。最近，糖苷水解酶家族的三个成员GH 115显示出从木聚糖的内部区域水解MeGlcA侧链，这是先前未描述的活性。在这里，我们发现，人类微生物群的主要成员，卵形拟杆菌，含有GH 115酶，BoAgu 115 A，它显示葡萄糖醛酸木聚糖α-（4-O-甲基）-葡萄糖醛酸酶活性。该酶对其中GlcA/MeGlcA修饰的木糖侧接一个或多个木糖残基的底物具有显著更高的活性。BoAgu 115 A的晶体结构揭示了一个四结构域蛋白，其中活性位点，包括一个口袋，邻接裂缝样结构，被安置在第二个结构域，采用TIM桶折叠。第三个结构域，一个五螺旋束，和C-末端β-夹心结构域进行链间接触，导致蛋白质二聚化。通过与开环形式的GlcA复合的酶的结构，结合诱变研究，鉴定了潜在的底物结合和催化显著的氨基酸。基于位于高度柔性环上的残基的催化重要性，需要酶经历实质性构象变化以与葡糖醛酸木聚糖形成生产性米氏复合物。
Background: The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown. Results: Bacteroides ovatus GH115 glucuronidase is a dimeric enzyme that contains a flexible active site pocket. Conclusion: The assembly of the catalytic apparatus of the glucuronidase requires substantial conformational changes. Significance: Conformational changes are highly unusual in glycoside hydrolases. The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.