Exploring novel strategies for depolymerization of plant cell-wall polysaccharides: from structure, function and rational design of glycosyl hydrolases to biological implications and potential biotechnological applications

Abstract

The plant cell wall is an important physical barrier to pathogens, and to chemical and physical stress for plants and it is essentially composed of polysaccharides of great biological and industrial importance. However, the challenge to deconstruct the plant cell wall into simple sugars is not a fully consolidated and economically viable process for many industrial applications as, for example, in the production of biofuels. Thus, develop and discover new strategies and processes biochemically more effective in dismantling the plant cell wall are of great economic interest. Furthermore, there is a strong biological appeal over the glycoside hydrolases (GHs) due to their direct involvement in a number of physio(patho)logical events including plant-pathogen interaction, the carbon cycle and homeostasis of plants. Despite great advances in the field Carbohydrate Enzymology, our knowledge is still limited on the vast universe of GHs. Only a very small fraction of the nature´s repertoire is known and this is evidenced by low rate of characterized emzymes in relation to the total of known sequences. Of the more than 330,000 GHs classified in CAZy database, only 2% have been biochemically studied and even less from a structural view. Among the various niches virtually unexplored, it highlights the broad and diverse GH arsenal of some plant pathogens and enzymes belonging to uncharacterized subfamilies. The bacterium that causes citrus canker (Xanthomonas axonopodis pv citri; Xac), for example, has in its genome more than 100 GH genes spread into 45 families, although it does not perform massive maceration of the plant cell wall. Compared to other species of the same genus as Xanthomonas albinieans (56 GHs, 32 families), there is an expansion in both the quantity and the diversity of GHs in Xac, suggesting an important role for these enzymes in their evolutive adaptation to citrus plants. However, the biochemical and physiological function of this vast enzymatic repertoire is still poorly understood and our research group has been a pioneer in demonstrating their biotechnological potential. In a recent study, we developed a highly efficient chimeric endoxylanase from the characterization and the rational redesign of a Xac exo-enzyme, which had its patent applied for industrial uses. In addition to the bias of functional and structural studies to GHs of certain species, a literature survey clearly shows the random and redundant way that these studies have been conducted within the various families of glycoside hydrolases. According to phylogenetic and similarity network analyses, a number of isofunctional clusters remain unknown at both functional and structural view. These isofunctional clusters, as demonstrated for the families 5, 13, 16 and 43, exhibit a range of activities and modes of action. However, the characterization of few members of each family is insufficient to reveal all functional diversity contained in the subfamilies, which usually have sequence identity lower than 30% of each other. Therefore, in this project we aim to explore and understand, at the mechanistic level, the functional diversity of GHs of unexplored niches such as the Xac pathogen and uuncharacterized subfamilies of the families 5 and 128. To accomplish with these objectives, we will use a multidisciplinary approach that integrates evolutionary, biochemical and structural studies to provide results that potentially will reveal novel strategies for depolymerization of plant cell wall and will generate instrumental data for the rational and optimized use of GHs for industrial purposes. Furthermore, using Xac pathogen as a model organism, we expect to better understand the role of GHs in the complex mechanisms mediating plant-pathogen interactions, thus contributing to assess the potential of these metabolic routes as targets for the development of molecular strategies employed to combat agricultural diseases economically relevant to the country. (AU)