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On the functional characterization of lytic polysaccharide monooxygenases (LPMOs).

ABSTRACT: Lytic polysaccharide monooxygenases (LPMOs) are abundant in nature and best known for their role in the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. LPMO activity requires an oxygen co-substrate, which was originally thought to be O2, but which may also be H2O2. Functional characterization of LPMOs is not straightforward because typical reaction mixtures will promote side reactions, including auto-catalytic inactivation of the enzyme. For example, despite some recent progress, there is still limited insight into the kinetics of the LPMO reaction. Recent discoveries concerning the role of H2O2 in LPMO catalysis further complicate the picture. Here, we review commonly used methods for characterizing LPMOs, with focus on benefits and potential pitfalls, rather than on technical details. We conclude by pointing at a few key problems and potential misconceptions that should be taken into account when interpreting existing data and planning future experiments.

SUBMITTER: Eijsink VGH

PROVIDER: S-EPMC6423801 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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On the functional characterization of lytic polysaccharide monooxygenases (LPMOs).

Eijsink Vincent G H VGH Petrovic Dejan D Forsberg Zarah Z Mekasha Sophanit S Røhr Åsmund K ÅK Várnai Anikó A Bissaro Bastien B Vaaje-Kolstad Gustav G

Biotechnology for biofuels 20190319

Lytic polysaccharide monooxygenases (LPMOs) are abundant in nature and best known for their role in the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. LPMO activity requires an oxygen co-substrate, which was originally thought to be O2, but which may also be H2O2. Functional characterization of LPMOs is not straightforward because typical reaction mixtures will promote side reactions, including auto-catalytic inactivation of th ...[more]

PMID: 30923566

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Project description:The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO2]+ complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H2O2, which was recently suggested to be the true co-substrate, rather than O2. The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol-1, compared to the much higher barrier (156 kJ mol-1) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.

Dataset Information

On the functional characterization of lytic polysaccharide monooxygenases (LPMOs).

Publications

On the functional characterization of lytic polysaccharide monooxygenases (LPMOs).

Similar Datasets

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