Freelove A C, Bolam D N, White P, Hazlewood G P, Gilbert H J
Laboratory of Molecular Enzymology, The Babraham Institute, Babraham Hall, Babraham, Cambridge CB2 4AT, United Kingdom.
J Biol Chem. 2001 Nov 16;276(46):43010-7. doi: 10.1074/jbc.M107143200. Epub 2001 Sep 17.
The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Anaerobic fungi that colonize herbivores are the most efficient plant cell wall degraders known, and this activity is vested in a high molecular weight complex that binds tightly to the plant cell wall. To investigate whether plant cell wall attachment is mediated by noncatalytic proteins, a cDNA library of the anaerobic fungus Piromyces equi was screened for sequences that encode noncatalytic proteins that are components of the cellulase-hemicellulase complex. A 1.6-kilobase cDNA was isolated encoding a protein of 479 amino acids with a M(r) of 52548 designated NCP1. The mature protein had a modular architecture comprising three copies of the noncatalytic dockerin module that targets anaerobic fungal proteins to the cellulase-hemicellulase complex. The two C-terminal modules of NCP1, CBM29-1 and CBM29-2, respectively, exhibit 33% sequence identity with each other but have no homologues in protein data bases. A truncated form of NCP1 comprising CBM29-1 and CBM29-2 (CBM29-1-2) and each of the two individual copies of CBM29 bind primarily to mannan, cellulose, and glucomannan, displaying the highest affinity for the latter polysaccharide. CBM29-1-2 exhibits 4-45-fold higher affinity than either CBM29-1 or CBM29-2 for the various ligands, indicating that the two modules, when covalently linked, act in synergy to bind to an array of different polysaccharides. This paper provides the first report of a CBM-containing protein from an anaerobic fungal cellulase-hemicellulase complex. The two CBMs constitute a novel CBM family designated CBM29 whose members exhibit unusually wide ligand specificity. We propose, therefore, that NCP1 plays a role in sequestering the fungal enzyme complex onto the plant cell wall.
微生物植物细胞壁水解酶对光合固定碳的循环利用是一个基本的生物学过程，它是主要地球化学循环之一的组成部分，此外还具有巨大的工业潜力。作用于植物细胞壁的酶系统包含非催化性碳水化合物结合模块（CBMs），这些模块介导与这种复合结构的附着，并在使水解过程最大化方面发挥关键作用。定殖于食草动物体内的厌氧真菌是已知最有效的植物细胞壁降解者，这种活性存在于一种与植物细胞壁紧密结合的高分子量复合物中。为了研究植物细胞壁的附着是否由非催化性蛋白质介导，对厌氧真菌埃氏梨形霉的cDNA文库进行筛选，以寻找编码作为纤维素酶 - 半纤维素酶复合物组分的非催化性蛋白质的序列。分离出一个1.6千碱基的cDNA，其编码一种由479个氨基酸组成、分子量为52548的蛋白质，命名为NCP1。成熟蛋白具有模块化结构，包含三个非催化性dockerin模块拷贝，该模块可将厌氧真菌蛋白靶向纤维素酶 - 半纤维素酶复合物。NCP1的两个C末端模块，即CBM29 - 1和CBM29 - 2，彼此间分别具有33%的序列同一性，但在蛋白质数据库中没有同源物。包含CBM29 - 1和CBM29 - 2的NCP1截短形式（CBM29 - 1 - 2）以及CBM29的两个单独拷贝中的每一个主要与甘露聚糖、纤维素和葡甘露聚糖结合，对后一种多糖显示出最高亲和力。CBM29 - 1 - 2对各种配体的亲和力比CBM29 - 1或CBM29 - 2高4 - 45倍，表明这两个模块共价连接时协同作用以结合一系列不同的多糖。本文首次报道了来自厌氧真菌纤维素酶 - 半纤维素酶复合物的含CBM的蛋白质。这两个CBM构成了一个新的CBM家族，命名为CBM29，其成员表现出异常广泛的配体特异性。因此，我们认为NCP1在将真菌酶复合物螯合到植物细胞壁上发挥作用。
