A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan
A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan
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DOI：
10.1074/jbc.ra119.009523
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发表时间：
2019-11-15
影响因子：
4.8
通讯作者：
Wilkens, Casper
中科院分区：
文献类型：
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作者：
Holck, Jesper;Fredslund, Folmer;Wilkens, Casper
阿魏酸酯酶(EC 3.1.1.73)属于糖酯酶家族1(CE1)，是阿拉伯木聚糖中阿魏酸(FA)和阿拉伯糖链之间的酯键水解酶。最近，在元基因组学研究中发现的一些CE1酶被预测含有48家族碳水化合物结合模块(CBM48)，这是一个与淀粉结合相关的CBM家族。其中两个CE1，污水处理污泥(Wts)Fae1A和wtsFae1B，具有同源的CBM48结构域，是阿魏酸酯酶，wtsFae1A与阿拉伯木聚糖结合。在这里，我们发现wtsFae1B也能与阿拉伯木聚糖结合，但两者都不能结合淀粉。表面等离子体共振分析表明，wtsFae1B对木糖的K-d为14.8微米，不与淀粉模拟物、β-环糊精或麦芽六糖结合。有趣的是，在没有CBM48结构域的情况下，wtsFae1A和wtsFae1B的CE1区域不能与阿拉伯木聚糖结合，也不能催化阿拉伯木聚糖释放FA。在没有CBM48的情况下，β-D-1，4-木聚糖酶能够使CE1结构域介导的FA从阿拉伯木聚糖中释放出来，这表明CBM48对多糖上的CE1活性是必不可少的。WtsFae1A(1.63埃分辨率)和wtsFae1B(1.98埃)的晶体结构表明，这两种蛋白质都是折叠蛋白质，都含有结构保守的氢键，锁定了CBM48相对于CE1结构域的位置。WtsFae1A对接表明，这两种酶都在CE1-CBM48结构域界面的裂隙中容纳了阿拉伯木聚糖主干。在这个裂隙上的结合似乎使CE1在聚合阿拉伯木聚糖上具有活性，说明CBM48结构域在调节阿拉伯木聚糖方面发挥了意想不到的关键作用。
Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's K-d for xylohexaose is 14.8 mu m and that it does not bind to starch mimics, beta-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a beta-D-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 angstrom resolution) and wtsFae1B (1.98 angstrom) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.