Dissecting the role of carbohydrate binding modules in plant cell wall degradation

植物细胞壁含有地球上最丰富的有机碳来源，其微生物对其组成糖的降解具有相当大的生物学和工业重要性。事实上，光合作用固定碳的循环对于草食动物的营养、陆地和海洋微生物生态系统的维持以及几种植物病原体对宿主的入侵至关重要。虽然攻击植物细胞壁的酶已广泛用于造纸、纺织、洗涤剂和食品(动物和人类)等几个以生物技术为基础的行业，但这些生物催化剂的主要应用是将植物生物质转化为生物乙醇和其他形式的能源。植物细胞壁主要由一系列不同的多糖组成，这些多糖通过复杂的氢键网络相互作用。它高度抵抗生物降解，因为多糖之间的广泛相互作用极大地限制了攻击这种复合结构的糖苷水解酶和酯酶电池的获得。微生物植物细胞壁水解酶具有复杂的分子结构，其中催化模块被附加到一个或多个非催化碳水化合物结合模块(CBM)上。大量的体外研究表明，CBMS通过与不溶性纯化的植物结构多糖结合，使同源酶与其目标底物紧密而持久地结合，从而在一定程度上克服了“可及性问题”，从而显著增强了催化作用。有趣的是，申请者最近的研究表明，CBM在结构上不同，但与纯化的配体具有相同的特异性，在完整植物细胞壁的背景下识别其目标多糖的能力显示出非常显著的差异。植物中配基识别的这种变化可能反映了目标多糖与细胞壁的其他成分的相互作用。因此，我们认为不同的CBM结合位点的拓扑结构适合于在特定生物的特定细胞类型中识别它们的目标多糖。到目前为止，对CBM在酶作用中的功能重要性的分析仅限于探索它们相对于纯化的底物或简单的、高度加工的复合材料的作用。鉴于CBM在植物中发挥着复杂的靶向作用，这些模块在降解完整的植物细胞壁方面的功能重要性目前尚不清楚。虽然很明显，这些模块将通过加强酶底物的接触来增强催化作用，但它们也可能在组装显示互补活性的糖苷水解酶和/或酯酶方面发挥作用，从而加强这些生物催化剂之间的协同作用。这一提议将检验这样一种假设，即细菌CBM多样性的生物学基础是：1)使同源酶能够访问位于不同植物细胞壁的目标底物，聚合物的背景会有所不同；以及2)将具有互补活性的酶招募到植物细胞壁的区域，在那里生物催化剂之间的协同作用最大限度地促进降解过程。该研究方案具有基本的生物学意义，因为这一过程是草食动物、植物和微生物之间营养循环的组成部分。从工业的角度来看，这些数据将提供信息并指导旨在产生新的糖苷水解酶和酯酶的战略，这些酶对植物细胞壁表现出更高的活性。这些酶在植物生物质的生物技术开发中将具有相当大的工业用途，特别是在生产生物乙醇方面，但也在造纸、动物和人类饲料、洗涤剂和纺织部门。

The plant cell wall comprises the most abundant source of organic carbon on the planet and its microbial degradation to its constituent sugars is of considerable biological and industrial importance. Indeed, the recycling of photosynthetically fixed carbon is critical to herbivore nutrition, the maintenance of terrestrial and marine microbial ecosystems and host invasion by several phytopathogens. While the enzymes that attack the plant cell wall are already widely used in several biotechnology-based industries including the paper, textile, detergent and food (animal and human) sectors, the major application of these biocatalysts is the conversion of plant biomass into bio-ethanol and other forms of energy. The plant cell wall comprises predominantly of an array of different polysaccharides that interact with each other through complex hydrogen bonding networks. It is highly recalcitrant to biological degradation as the extensive interactions between the polysaccharides greatly restrict access to the battery of glycoside hydrolases and esterases that attack this composite structure. Microbial plant cell wall hydrolases display complex molecular architectures in which the catalytic module is appended to one or more non-catalytic carbohydrate binding modules (CBMs). Numerous in vitro studies have shown that by binding to insoluble purified plant structural polysaccharides, CBMs bring the cognate enzyme into intimate and prolonged association with their target substrate resulting in a significant potentiation of catalysis as, to some extent, they overcome the 'accessibility problem'. Intriguingly, recent studies by the applicants have shown that CBMs, which are structurally distinct but exhibit the same specificities against purified ligands, display highly significant differences in their capacity to recognise their target polysaccharides within the context of the complete plant cell wall. This variation in ligand recognition in planta likely reflects the interaction of the target polysaccharides with other components of the cell wall. Thus, we propose that the topology of the binding sites of different CBMs are adapted to recognize their target polysaccharides in specific cell types of specific organisms. To date the analysis of the functional importance of CBMs in enzyme action has been limited to exploring their role against purified substrates or simple, highly processed, composites. In view of the complex targeting role CBMs play in planta, the functional importance of these modules in degrading intact plant cell walls is currently unclear. While it is apparent that these modules will increase catalysis by enhancing enzyme substrate contact, they may also play a role in assembling glycoside hydrolases and/or esterases that display complementary activities into juxtapositions in the cell wall thereby potentiating the synergistic interactions between these biocatalysts. This proposal will test the hypothesis that the biological rationale for the diversity of bacterial CBMs is to 1) enable the cognate enzymes to access their target substrates located in different plant cell walls, where the context of the polymer will vary; and 2) to recruit enzymes with complementary activities to regions of the plant cell wall where the synergistic interactions between the biocatalysts maximise the degradative process. The research programme is of fundamental biological importance as the process is integral to the cycling of nutrients between herbivores, plants and microbes. From an industrial perspective the data will inform and direct strategies designed to generate novel glycoside hydrolases and esterases that display increased activity against plant cell walls. These enzymes would have considerable industrial utility in the biotechnological exploitation of plant biomass, particularly in the generation of bio-ethanol, but also in the paper, animal and human feed, detergent and textile sectors.