The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases.
The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases.
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DOI：
10.1074/jbc.m115.702365
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发表时间：
2016-04-01
期刊：
影响因子：
--
通讯作者：
Gilbert HJ
中科院分区：
文献类型：
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作者：
Crouch LI;Labourel A;Walton PH;Davies GJ;Gilbert HJ
木质纤维生物质是一种可持续的工业底物。铜依赖的裂解多糖单加氧酶(LPMOS)有助于木质纤维素的降解，提高生物燃料的生产效率。LPMO可以包含非催化碳水化合物结合模块(CBM)，但对它们在这些酶活性中的作用知之甚少。在这里，我们探讨了CBMS在LPMO功能中的重要性。Cellulomonas FIMI的CfLPMO10和TbLPMO10这两个单加氧酶的CfLPMO10和TbLPMO10家族的CBM被缺失和/或替换为来自其他蛋白质的CBM。数据表明，CBMS可以增强LPMO活性，也可以令人惊讶地抑制LPMO活性，而且这些影响既是酶特异性的，也是底物特异性的。去除天然CBM或引入CtCBM3a，几乎完全丧失了LPMOS对纤维素底物的催化活性。去除CBM的有害影响可能反映了酶在底物表面长时间呈现对于有效催化活性的重要性，因为只有附着在CBM上的LPMO才与纤维素紧密结合。CtCBM3a的负面影响与该结合模块增强包括纤维素酶在内的一系列糖苷水解酶活性的能力形成鲜明对比。从CfLPMO10中删除内源CBM或将来自日本细胞弧菌LPMO10B的10族CBM导入TbLPMO10中，都会影响非氧化产物的生成，表明CBM可以调节LPMOs的作用方式。这项研究表明，经过工程改造的LPMO-CBM杂化材料可以显示出增强的工业相关氧合作用。
Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.