Gabriel Paës

The Structure of the Complex between a Branched Pentasaccharide and Thermobacillus Xylanilyticus Gh-51 Arabinofuranosidase Reveals Xylan-Binding Determinants and Induced Fit.

The crystal structure of the family GH-51 alpha- l-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural substrate xylan. The overall structure shows the two characteristic GH-51 domains: a catalytic domain that is folded into a (beta/alpha) 8-barrel and a C-terminal domain that displays jelly roll architecture. The pentasaccharide is bound in a groove on the surface of the enzyme, with the mono arabinosyl branch entering a tight pocket harboring the catalytic dyad. Detailed analyses of both structures and comparisons with the two previously determined structures from Geobacillus stearothermophilus and Clostridium thermocellum reveal important details unique to the Thermobacillus xylanilyticus enzyme. In the absence of substrate, the enzyme adopts an open conformation. In the substrate-bound form, the long loop connecting beta-strand 2 to alpha-helix 2 closes the active site and interacts with the substrate through residues His98 and Trp99. The results of kinetic and fluorescence titration studies using mutants underline the importance of this loop, and support the notion of an interaction between Trp99 and the bound substrate. We suggest that the changes in loop conformation are an integral part of the T. xylanilyticus alpha- l-arabinofuranosidase reaction mechanism, and ensure efficient binding and release of substrate.

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (Kd) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

Characterization of arabinoxylan/cellulose nanocrystals gels to investigate fluorescent probes mobility in bioinspired models of plant secondary cell wall.

Biomass from lignocellulose (LC) is a highly complex network of cellulose, hemicellulose, and lignin, which is considered to be a sustainable source of fuels, chemicals and materials. To achieve an environmental friendly and efficient LC upgrading, a better understanding of the LC architecture is necessary. We have devised some LC bioinspired model systems, based on arabinoxylan gels, in which mobility of dextrans and BSA grafted with FITC has been studied by FRAP. Our results indicate that the probes diffusion is more influenced by their hydrodynamic radius than by the gel mesh size. The addition of some cellulose nanocrystals (CNCs) decreases polymer chain mobility and has low effect on the probes diffusion, suggesting that the gels are better organized in the presence of CNCs, as shown by rheological measurements and scanning electronic microscopy observations. This demonstrates that the FRAP analysis can be a powerful tool to screen the architecture of LC model systems.

Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review

Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

Thumb-loops up for catalysis: a structure/function investigation of a functional loop movement in a GH11 xylanase

Dynamics is a key feature of enzyme catalysis. Unfortunately, current experimental and computational techniques do not yet provide a comprehensive understanding and description of functional macromolecular motions. In this work, we have extended a novel computational technique, which combines molecular modeling methods and robotics algorithms, to investigate functional motions of protein loops. This new approach has been applied to study the functional importance of the so-called thumb-loop in the glycoside hydrolase family 11 xylanase from Thermobacillus xylanilyticus (Tx-xyl). The results obtained provide new insight into the role of the loop in the glycosylation/deglycosylation catalytic cycle, and underline the key importance of the nature of the residue located at the tip of the thumb-loop. The effect of mutations predicted in silico has been validated by in vitro site-directed mutagenesis experiments. Overall, we propose a comprehensive model of Tx-xyl catalysis in terms of substrate and product dynamics by identifying the action of the thumb-loop motion during catalysis.

Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.

Knowledge book on lignocellulose deconstruction: an INRA project to identify key actions in research on biorefineries

The existence of pilot and industrial scale biorefineries worldwide demonstrates the technical and economic feasibility of fractionating lignocellulose (LC) for chemistry and energy. This raises new questions about the biomass supply, management of its quality and about the elementary step combination in processes, to choose which compound will be main- or co-products from plant biomass. Integrated and systemic approaches are requested to invent and/or to improve the biotechnical fractionation of LCs and there is a need to collect and correlate the existing knowledge in a structured way, to gain a better insight of the overall process. Building such a knowledge representation is important for scientists, research institutes, universities and industries, as it will give a shared description of the knowledge in that field, that will further facilitate its diffusion, re-use, review, reassessment and updating with new findings. Practically, an extensive literature has been published in the past five years on the biorefinery of LC, focusing mostly on the saccharification of polysaccharides (cellulose, hemicellulose). As a consequence, most of the data available results from biochemical and physicochemical analyzes from several processing chains, which combine different modes of physical pretreatments and/or chemical typologies of variables biomass and/or various hydrolytic enzyme cocktails. That is why a project for development of a hypertext electronic Knowledge Book on LignoCellulose DeConstruction (KB-LCDC) was initiated by the French National Institute for Agricultural Research (INRA) with two main goals: i/ to elicit the available knowledge from various sources, more specifically related to the enzymatic hydrolysis of wheat straw into glucose, ii/ to represent the knowledge and implement it into a web-based format of Knowledge Book (KB) taking into account the overall saccharification process. The knowledge was first elicited by means of semi-structured interviews with a group of six experts working in several INRA research Units, and involved in the Institute’s biomass transformation network. Concomitantly, the collating of data and knowledge from “grey-” and peer reviewed- literature was also done. Then, our approach consists in building a knowledge book (KB) whose pages are formatted concept maps (Cmap) and technical sheets that are connected by hypertext links. A Cmap is a semantic graph where nodes represent concepts that are connected by arcs expressing relationships between them. A formatted Cmap answers a specific question about one central concept (for instance: what is the impact of the pretreatment on the reactivity of biomass ? How does enzyme diffuse into the lignocellulose ?). Hyperlinks existing between Cmaps and technical sheets form a network of knowledge, into which the user can navigate, to find relevant answers, but also associated concepts. Hyperlinks can also link Cmaps or technical sheet to an Internet page, scientific article and any document selected to illustrate the reality of a concept. Up to nine knowledge areas have been identified so far, among them: biomass pretreatment; separation methods; enzyme cocktails; substrate reactivity, hydrolysis mechanisms. A global representation of the overall process from wheat straw to glucose, based on the individual Cmaps, has been built. It includes a static structural view (environment and reactivity of the media, encompassing the cell wall); a dynamic view (hierarchy of the different sub-processes at work) and a functional view (description of the elementary steps and how they are organized in time). In the frame of the LBT III congress, the practical structuration of the knowledge and the original version of the KB will be disclosed. The potential development and use of this new approach for the representation of biotechnology processes applied to LC will be discussed (process workflow; unlocking cell wall recalcitrance; strategic roadmaps).