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

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.

The primary objective of this research programme is to investigate the mechanism by which carbohydrate binding modules (CBMs) potentiate the activity of glycoside hydrolases against complete plant cell walls. We propose that the wide range of CBMs present in bacterial enzymes maximise the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall but also increase the range of plant cell walls that can be degraded. In addition to maximising substrate access we also propose that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plant cell wall thereby maximising the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase access of closely associated polymers to enzyme attack. The research programme will contain two major components; 1) the construction of hybrid enzymes containing numerous combinations of catalytic domains fused to CBMs; 2) analysis of the capacity of these enzymes to attack plant cell walls using both an immunohistochemical approach to dissect the spatial degradation of the plant cell wall in harness with a biochemical approach to quantify and identify the products released from these composite structures. Appropriate CBMs and the catalytic domains of glycoside hydrolases and esterases will be constructed and the biochemical and regio-selectivity of cell wall degradation will be evaluated. These studies will investigate the functional significance of CBMs that bind to crystalline cellulose, amorphous cellulose, xylan, xyloglucan and the reducing end of polysaccharide chains. The experiments will address key questions regarding the capacity of CBMs to not only target hydrolases to the plant cell wall but also to potentiate the synergy between the enzymes, which is central to the degradation of highly complex composite structures. Joint with BB/015190/1