Structure and function of lytic polysaccharide monooxygenases (LPMOS)and other redox enzymes involved in biomass processing

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has revolutionized our understanding of biomass conversion in Nature and has been instrumental for the development of economically sustainable lignocellulose biorefineries. LPMOs are mono-copper redox enzymes that attack the most recalcitrant parts of biopolymers such as crystalline cellulose and chitin. LPMOs employ the power of redox chemistry to cleave glycosidic bonds that are not easily cleaved by hydrolytic enzymes. By doing so, they make the substrate more tractable to the action of canonical enzymes such as endo- and exo-cellulases. LPMOs are abundant in Nature, for example in the secretomes of wood-decaying fungi. Despite their importance in both Nature and the biorefinery, several aspects of these intriguing enzymes remain unclear. The catalytic mechanism of LPMOs is of particular importance because the enzymes display a unique active site architecture that is employed to catalyze a challenging chemical reaction on a substrate that is embedded in a crystalline lattice. Deeper insight into this mechanism may have wide-reaching consequences, not only for biomass processing but also, perhaps, in developing enzymatic or other catalytic systems for difficult reactions, such as controlled oxidation of methane and other alkanes. Using a variety of experimental approaches, we are studying LPMO function, addressing issues such as the structural basis of oxidative regio-selectivity and substrate specificity, routes and mechanisms for electron delivery, the roles of appended carbohydrate-binding domains, and the determinants of catalytic activity and stability. Knowledge gained from these fundamental studies is being used to optimize biomass conversion processes, whereas translation of this knowledge to other fields is also being explored. In this presentation, I will review recent work in the field and present our latest results. Special attention will be paid to recent research in our group that has led to the proposal that LPMOs may not be true monooxygenases. Based on our recent results, we have proposed that hydrogen peroxide, rather than molecular oxygen, is the preferred co-substrate of LPMOs. While this paradigm-shattering proposal may not yet have found wide acceptance in the field, we have already demonstrated that the controlled administration of hydrogen peroxide during biomass degradation by LPMO-containing commercial cellulolytic enzyme cocktails leads to drastically improved LPMO activity and more efficient saccharification. These recent findings also shed new light on the interplay between LPMOs and other redox enzymes in the secretomes of biomass degrading microorganisms

A fungal family of lytic polysaccharide monooxygenase-like copper proteins

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation

La partition Hydrolyse / Transglycosylation chez les Glycoside Hydrolases : Proposition d’une hypothèse de synthèse à travers l’évolution moléculaire d’une α-L-arabinofuranosidase de la famille GH51 vers les premières transarabinofuranosylases de type non-Leloir

Widening the spectrum of available compounds in the field of Glycosciences is of utmost importance for the entire biology community, because carbohydrates are determinants of a myriad of life-sustaining or threatening processes. The assembly, modification or deconstruction of complex carbohydrate-based structures mainly involves the action of enzymes, among which one can identify Carbohydrate Active enZymes (CAZymes). These enzymes form part of the CAZy database repertoire and include Glycoside Hydrolases (GHs), which are the biggest group of CAZymes, whose main role is to hydrolyze glycosidic linkages. However, some GHs also display the ability to perform synthesis (transglycosylation), an activity that mostly manifests itself as a minor one alongside hydrolysis, but which is the only activity displayed by a rather select group of GHs that are often called transglycosylases. Understanding how transglycosylases have resulted from the process of evolution is both intringuing and crucial, because it holds the key to the creation of tailored glycosynthetic enzymes that will revolutionize the field of glycosciences.In this thesis, an extensive review of relevant scientific literature that treats the different aspects of GH-catalyzed transglycosylation and glycosynthesis is presented, along with experimental results of work that has been performed on a family GH-51 α-L-arabinofuranosidase, a pentose-acting enzyme from Thermobacillus xylanilyticus (TxAbf). The conclusions of the literature are presented in the form of a hypothesis, which describes the molecular basis of the hydrolysis/transglycosylation partition and thus provides a proposal on how to engineer dominant transglycosylation activity in a GH. Afterwards, using a directed evolution approach, including random mutagenesis, semi-rational approaches, in silico predictions and recombination it has been experimentally possible to create the very first ‘non-Leloir’ transarabinofuranosylases. The mechanistic analysis of the resultant TxAbf mutants notably focusing on the hydrolysis/transglycosylation partition reveals that the results obtained are consistent with the initial hypothesis that was formulated on the basis of the literature review.To demonstrate the applicative value of the experimental work performed in this study, the TxAbf mutants were used to develop a chemo-enzymatic methodology that has procured a panel of well-defined furanosylated compounds. Enzyme-catalyzed transfer of arabinofuranosyl moities can be used to generate arabinoxylo-oligosaccharides (AXOS), but the design of non-natural oligosaccharides, such as galactofuranoxylo-oligosaccharides or arabinofuranogluco-oligosaccharides is also possible. Overall, the work presented constitutes the first steps towards the development of more sophiscated methodologies that will procure the means to synthesize artificial arabinoxylans, with a first proof of concept being presented at the very end of this manuscript.In the present context of the bioeconomy transition, which relies on technologies such as biorefining and green chemistry, it is expected that the glycosynthetic tools that have been developed in this work will be useful for the conversion of pentose sugars obtained from biomass. The synthesis of tailor-made arabinoxylo-oligo- and polysaccharides may lead to a variety of potential applications including the production of prebiotics, surfactants or bio-inspired materials and, more fundamentally, the synthesis of artificial models of plant cell wall.Élargir le répertoire de composés accessibles dans le domaine des Glycosciences est d’un intérêt majeur pour la communauté des biologistes du fait que ces composés, oligosaccharides et glyco-conjugués, sont impliqués dans diverses fonctions biologiques, aussi bien au niveau structurel, qu’énergétique voire même signalétique jouant un rôle primordial dans les interactions inter- ou intracellulaires. L’assemblage, la modification ou la déconstruction de ces glyco-structures complexes est possible grâce à l’action d’enzymes, parmi lesquelles l’on retrouve les CAZymes (Carbohydrate Active enZymes). Ces enzymes font partie du répertoire de la base de données CAZy, incluant les Glycoside Hydrolases (GHs) qui représentent le groupe le plus important et ayant pour fonction biologique principale l’hydrolyse des liens glycosidiques. Cependant, un certain nombre de GHs possède aussi la capacité de catalyser des réactions de synthèse (transglycosylation) en tant qu’activité secondaire mineure, voire en tant qu’activité principale pour un nombre restreint d’entre elles, qui sont alors appelées transglycosylases. Sachant que ces deux types de comportements peuvent se retrouver au sein d’une même famille de GH (donc étroitement liés sur le plan évolutif), la découverte et la compréhension des déterminants moléculaires qui ont été développés par les GHs au cours de leur évolution pour permettre cette partition d’activité, entre hydrolyse et transglycosylation, est d’une importance capitale pour le domaine de la synthèse chimio-enzymatique et des Glycosciences de manière plus générale.Ce travail de thèse décrit une proposition de synthèse pour apporter une réponse à cette question fondamentale via une revue critique de la littérature sur le sujet. Sur le plan expérimental, a été réalisée l’évolution moléculaire d’une enzyme spécifique des pentoses, l’α-L-arabinofuranosidase de Thermobacillus xylanilyticus (TxAbf) de la famille GH51, vers les premières transarabinofuranosylases de type ‘non-Leloir’. Cette évolution itérative a été développée en utilisant un panel d’outils d’ingénierie enzymatique combinant des approches aléatoire, semi-rationnelle, de prédiction in silico suivie de recombinaison dans un processus d’évolution dirigée global. Une analyse fine des mutants générés sur le plan mécanistique en lien avec la partition hydrolyse/transglycosylation mène à des conclusions en accord avec la proposition de synthèse issue de la revue de la littérature sur le sujet. Sur un plan plus appliqué, ces nouveaux biocatalyseurs ont ensuite été mis en oeuvre dans des voies de synthèse chimio-enzymatiques pour la préparation de composés furanosylés de structure contrôlée. Le transfert d’L-arabinofuranosyles permet la génération d’arabinoxylo-oligosaccharides (AXOS) ainsi que la conception d’oligosaccharides non naturels, tel que des galactofuranoxylo-oligosaccharides ou des arabinofuranogluco-oligosaccharides. Dans son ensemble, ce travail de recherche constitue les premières étapes clés du développement de méthodes de synthèse chimio-enzymatique plus élaborées pour la conception d’arabinoxylanes artificiels. Dans le contexte actuel de transition vers une bio-économie, reposant sur des concepts tels que ceux de la bioraffinerie ou de la chimie verte, nous espérons que les outils de glycosynthèse développés au cours de ces travaux trouveront leur application dans la valorisation des pentoses issus de la biomasse. La synthèse à-façon d’arabinoxylooligo- et polysaccharides présente nombre de valorisations possibles allant de la préparation de prébiotiques à la conception de matériaux bio-inspirés en passant par la synthèse de modèles de parois végétales

Lytic polysaccharide monooxygenases: enzymes for controlled and site-specific Fenton-like chemistry

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has profoundly changed our current understanding of enzymatic processes underlying the conversion of polysaccharides in the biosphere. LPMOs are truly unique enzymes, harboring a single copper atom in a solvent-exposed active site, allowing them to oxidize C-H bonds at the C1 and/or C4 carbon of glycosidic linkages found in recalcitrant, often crystalline polysaccharides such as cellulose and chitin. To catalyze this challenging reaction, LPMOs harness and control a powerful oxidative reaction that involves Fenton-like chemistry. In this essay, we first draw a brief portrait of the LPMO field, notably explaining the shift from the monooxygenase paradigm (i.e., using O2 as cosubstrate) to that of a peroxygenase (i.e., using H2O2). Then, we briefly review current understanding of how LPMOs generate and control a hydroxyl radical (HO•) generated through Cu(I)-catalyzed H2O2 homolysis, and how this radical is used to create the proposed Cu(II)-oxyl species, abstracting hydrogen atom of the C-H bond. We also point at the complexity of analyzing redox reactions involving reactive oxygen species and address potential deficiencies in the interpretation of existing LPMO data. Being the first copper enzymes shown to enable site-specific Fenton-like chemistry, and maybe not the only ones, LPMOs may serve as a blueprint for future research on monocopper peroxygenases

Structural diversity of lytic polysaccharide monooxygenases

acceptedVersio

Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

International audienceLytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where theycatalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance ofLPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies,with kcat/KmH2O2 values in the order of 105–106M−1 s−1. Besides providing crucial insight intothe cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

International audienceCarbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases

The rotamer of the second sphere histidine in AA9 Lytic polysaccharide monooxygenase is pH-dependent

International audienceLytic polysaccharide monooxygenases (LPMOs) catalyze a reaction that is crucial for the biological decomposition of various biopolymers and for the industrial conversion of plant biomass. Despite the importance of LPMOs, the exact molecular-level nature of the reaction mechanism is still debated today. Here, we investigated the pH-dependent conformation of a second-sphere histidine (His) that we call the stacking histidine, which is conserved in fungal AA9 LPMOs and is speculated to assist catalysis in several of the LPMO reaction pathways.Using constant-pH and accelerated molecular dynamics simulations, we monitored the dynamics of the stacking His in different protonation states for both the resting Cu(II) and active Cu(I) forms of two fungal LPMOs. Consistent with experimental crystallographic and neutron diffraction data, our calculations suggest that the side chain of the protonated and positively charged form is rotated out of the active site toward the solvent. Importantly, only one of the possible neutral states of histidine (HIE state) is observed in the stacking orientation at neutral pH or when bound to cellulose. Our data predict that, in solution, the stacking His may act as a stabilizer (via hydrogen bonding) of the Cu(II)-superoxo complex after the LPMO-Cu(I) has reacted with O 2 in solution, which, in fine, leads to H 2 O 2 formation. Also, our data indicate that the HIE-stacking His is a poor acid/base catalyst when bound to the substrate and, in agreement with the literature, may play an important stabilizing role (via hydrogen bonding) during the peroxygenase catalysis.Our study reveals the pH titration midpoint values of the pH-dependent orientation of the stacking His should be considered when modeling and interpreting LPMO reactions, whether it be for classical LPMO kinetics or in industry-oriented enzymatic cocktails, and for understanding LPMO behavior in slightly acidic natural processes such as fungal wood decay

Controlled depolymerization of cellulose by light-driven lytic polysaccharide oxygenases

Lytic polysaccharide (mono)oxygenases (LPMOs) perform oxidative cleavage of polysaccharides, and are key enzymes in biomass processing and the global carbon cycle. It has been shown that LPMO reactions may be driven by light, using photosynthetic pigments or photocatalysts, but the mechanism behind this highly attractive catalytic route remains unknown. Here, prompted by the discovery that LPMOs catalyze a peroxygenase reaction more efficiently than a monooxygenase reaction, we revisit these light-driven systems, using an LPMO from Streptomyces coelicolor (ScAA10C) as model cellulolytic enzyme. By using coupled enzymatic assays, we show that H2O2 is produced and necessary for efficient light-driven activity of ScAA10C. Importantly, this activity is achieved without addition of reducing agents and proportional to the light intensity. Overall, the results highlight the importance of controlling fluxes of reactive oxygen species in LPMO reactions and demonstrate the feasibility of light-driven, tunable enzymatic peroxygenation to degrade recalcitrant polysaccharides