Mimicking LPMO enzymes : a structural study into the importance of the histidine brace motif
(2025)
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- Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent metalloenzymes that oxidatively cleave polysaccharides, facilitating biomass degradation. Their conserved active site features a single copper atom coordinated in a T-shaped N₃ configuration, known as the histidine brace motif. Cellulose-active LPMOs have attracted significant interest due to their potential in second-generation biofuel and bio-based chemical production. Positioned at the enzyme surface, the LPMO active site enables oxidative cleavage of strong glycosidic C–H bonds (BDE > 100 kcal/mol), disrupting cellulose structure. When combined with cellulases, LPMOs form a synergistic enzymatic system capable of efficient cellulose breakdown. Despite extensive research, the origin of the histidine brace oxidative power and its precise mechanism of action remain elusive. Discerning the role of individual structural features, intrinsically linked to the enzyme’s architecture, remains a significant challenge. The use of small synthetic models is a strategy that turned out to be highly valuable and insightful in many enzymatic systems, including the LPMOs. The strong disparity between the accurate models reported in the literature makes the comparison and the identification of important features challenging. Plus, the second coordination sphere of the active site is often overlooked. This thesis aimed at developing a series of structurally accurate LPMO active site models and establishing a strong structure-property relationship. To do so, two main approaches were explored. The first one was based of the design of small synthetic models with systematic structural variations, such as the methylation of the imidazole, the chelate ring size, the nature and the connectivity of the heterocycle as well as the presence of an imine or amine function, while the second approach studied the influence of the site isolation and, to a lesser extent, the second coordination sphere on the copper centre properties via a supramolecular approach. In the small model approach, the differences in properties and reactivity appeared to be primarily due to the electronic differences impacted by the structural modifications. The larger flexibility brought by the six-membered chelate rings as well as the presence of a pyridine and a 2-connected imidazole seemed to be the most important structural features for the improvement of the oxidative power of the compounds, supposedly due to the increased π-acceptance character of the ligands. The catalytic and mechanistic studies supported a free radical mechanism being the main pathway for the PNPG degradation in the case of these complexes. The amide-based ligands suggested to self-assemble in solution into a dimeric Cu2L2 structure, and generate the histidine brace motif with a primary amine and two disconnected imidazole displayed improved reactivity. Despite its known importance, control of the metal second environment and second coordination sphere is often neglected due to synthetic challenges. To tackle this problem, two supramolecular strategies were explored to physically isolate the models and modify their environment through non-covalent interactions. The first approach consisted of the functionalization of a model with an adamantane anchor group, known to interact with many macrocycles. The catalytic assays showed a reduced lag time when the complex was used in the presence of the macrocycles, emphasizing the impact of the host-guest adduct on the copper properties. The second approach consisted of the encapsulation of the small synthetic models inside the porous framework of zeolites, which was confirmed by a series of characterizations. While the influence of the site isolation on the models’ structural parameters and properties remains obscure, the zeolite catalysts displayed encouraging oxidative reactivity. This thesis established the beginning of a clear structure–property relationship for LPMO active site models and highlighted the importance of the second coordination sphere to modulate their properties.
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Gilbert, U. (2025). Mimicking LPMO enzymes : a structural study into the importance of the histidine brace motif.