Degradation of cellulose substrates by cellulosome chimeras -: Substrate targeting versus proximity of enzyme components
Degradation of cellulose substrates by cellulosome chimeras -: Substrate targeting versus proximity of enzyme components
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DOI：
10.1074/jbc.m207672200
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发表时间：
2002-12-20
影响因子：
4.8
通讯作者：
Bélaïch, JP
中科院分区：
文献类型：
--
作者：
Fierobe, HP;Bayer, EA;Bélaïch, JP
构建了75种不同嵌合纤维素体的文库，作为我们先前描述的用于产生模型功能复合物的方法的延伸（Fierobe，H. P.，Mechaly，A.，Tardif角，Belaich，A.，拉梅德河Shoham，Y.，Belaich，J. P.，Bayer，E. A（2001）J.BioLChem.276，21257 -21261），基于高亲和力的物种特异性粘附素-锚定蛋白相互作用。每个复合物含有三种蛋白质组分：（i）嵌合支架蛋白，其具有任选的纤维素结合模块和两个趋异特异性的粘附素，和（ii）两个纤维素酶，每个纤维素酶具有与趋异粘附素之一互补的锚定蛋白。使用不同类型的纤维素底物测定所得三元复合物的活性。纤维素分解酶的多纤维素酶体嵌合体的组织导致在特征性的高活性的柠檬酸底物，而多纤维素酶体嵌合体表现出很少或没有优势，超过自由酶系统的易处理的基板上。在柠檬酸纤维素上，支架蛋白上的纤维素结合结构域和所得复合物上的酶接近性的存在几乎同样有助于它们对底物的升高作用。然而，对于某些酶对，一种效应似乎比另一种效应占主导地位。结果还表明，基板的结晶度不一定是一个功能，但反映了反应位点的整体可及性。
A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Belaich, A., Lamed, R., Shoham, Y., Belaich, J.-P., and Bayer, E. A (2001) J. BioL Chem. 276,21257-21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.