Abstract
Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.
Highlights
IntroductionThe Lytic polysaccharide monooxygenases (LPMOs) active site is composed by a copper atom coordinated equatorially by the imidazole nitrogen atoms of two histidines, one of these being the N-terminal residue, and the nitrogen atom of the terminal amine, in a so-called histidine brace motif.[4] When copper is in the oxidized Cu(II) state, the coordination is completed by the oxygen atoms of a conserved Tyr residue and a water molecule
The active site model includes a greater number of atoms, compared to those used for previously reported computational investigations of the Lytic polysaccharide monooxygenases (LPMOs) mechanism
In the case of LPMOs, a very recent work from Span et al revealed the crucial role played by secondary coordination sphere in a fungal LPMO: in particular, they identified His and Gln amino acids, belonging to a H-bond network conserved in all fungal LPMO families, as important residues for oxygen activation.[48]
Summary
The LPMO active site is composed by a copper atom coordinated equatorially by the imidazole nitrogen atoms of two histidines, one of these being the N-terminal residue, and the nitrogen atom of the terminal amine, in a so-called histidine brace motif.[4] When copper is in the oxidized Cu(II) state, the coordination is completed by the oxygen atoms of a conserved Tyr residue and a water molecule Based on their substrate specificity and on the host organism, LPMOs are currently classified into four Auxiliary Activity (AA) families in the Carbohydrate-Active enZyme (CAZy) database:[15] cellulose- and hemi-cellulose active fungal LPMOs (forming the AA9 family); chitin- and cellulose-active bacterial LPMOs (AA10); chitin-active fungal LPMOs (AA11) and fungal starchdegrading LPMOs (AA13). Major insights were gained thanks to the crystal structure of an LPMO9 with bound oligosaccharide,[20] revealing that hydrogen bonds between surface exposed Asn and His residues play a crucial role in the interaction with the substrate, together with an electrostatic interaction between the N-terminal His[1] imidazole and the ring oxygen of the sugar occupying the +1 site
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