The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.
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ABSTRACT:
SUBMITTER: Frandsen KE
PROVIDER: S-EPMC4817220 | biostudies-literature | 2016 Apr
REPOSITORIES: biostudies-literature
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The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.
Frandsen Kristian E H KE Simmons Thomas J TJ Dupree Paul P Poulsen Jens-Christian N JC Hemsworth Glyn R GR Ciano Luisa L Johnston Esther M EM Tovborg Morten M Johansen Katja S KS von Freiesleben Pernille P Marmuse Laurence L Fort Sébastien S Cottaz Sylvain S Driguez Hugues H Henrissat Bernard B Lenfant Nicolas N Tuna Floriana F Baldansuren Amgalanbaatar A Davies Gideon J GJ Lo Leggio Leila L Walton Paul H PH
Nature chemical biology 20160229 4
Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination ...[more]
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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.
| S-EPMC5935029 | biostudies-literature
Project description:Lytic polysaccharide monooxygenase (LPMO) enzymes have attracted considerable attention owing to their ability to enhance polysaccharide depolymerization, making them interesting with respect to production of biofuel from cellulose. LPMOs are metalloenzymes that contain a mononuclear copper active site, capable of activating dioxygen. However, many details of this activation are unclear. Some aspects of the mechanism have previously been investigated from a computational angle. Yet, either these studies have employed only molecular mechanics (MM), which are inaccurate for metal active sites, or they have described only the active site with quantum mechanics (QM) and neglected the effect of the protein. Here, we employ hybrid QM and MM (QM/MM) methods to investigate the first steps of the LPMO mechanism, which is reduction of CuII to CuI and the formation of a CuII-superoxide complex. In the latter complex, the superoxide can bind either in an equatorial or an axial position. For both steps, we obtain structures that are markedly different from previous suggestions, based on small QM-cluster calculations. Our calculations show that the equatorial isomer of the superoxide complex is over 60 kJ/mol more stable than the axial isomer because it is stabilized by interactions with a second-coordination-sphere glutamine residue, suggesting a possible role for this residue. The coordination of superoxide in this manner agrees with recent experimental suggestions.
| S-EPMC6641039 | biostudies-literature
Project description:BackgroundLytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC).ResultsThe fungal LPMO from AA9 family (PaLPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with PaLPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC.ConclusionsThis study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.
| S-EPMC6589874 | biostudies-literature
Project description:BackgroundLytic polysaccharide monooxygenases are important enzymes for the decomposition of recalcitrant biological macromolecules such as plant cell wall and chitin polymers. These enzymes were originally designated glycoside hydrolase family 61 and carbohydrate-binding module family 33 but are now classified as auxiliary activities 9, 10 and 11 in the CAZy database. To obtain a systematic analysis of the divergent families of lytic polysaccharide monooxygenases we used Peptide Pattern Recognition to divide 5396 protein sequences resembling enzymes from families AA9 (1828 proteins), AA10 (2799 proteins) and AA11 (769 proteins) into subfamilies.ResultsThe results showed that the lytic polysaccharide monooxygenases have two conserved regions identified by conserved peptides specific for each AA family. The peptides were used for in silico PCR discovery of the lytic polysaccharide monooxygenases in 79 fungal and 95 bacterial genomes. The bacterial genomes encoded 0-7 AA10s (average 0.6). No AA9 or AA11 were found in the bacteria. The fungal genomes encoded 0-40 AA9s (average 7) and 0-15 AA11s (average 2) and two of the fungi possessed a gene encoding a putative AA10. The AA9s were mainly found in plant cell wall-degrading asco- and basidiomycetes in agreement with the described role of AA9 enzymes. In contrast, the AA11 proteins were found in 36 of the 39 ascomycetes and in only two of the 32 basidiomycetes and their abundance did not correlate to the degradation of cellulose and hemicellulose.ConclusionsThese results provides an overview of the sequence characteristics and occurrence of the divergent AA9, AA10 and AA11 families and pave the way for systematic investigations of the of lytic polysaccharide monooxygenases and for structure-function studies of these enzymes.
| S-EPMC4424831 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.
| S-EPMC5223172 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes for the degradation of recalcitrant polysaccharides such as chitin and cellulose. Unlike classical hydrolytic enzymes (cellulases), LPMOs catalyze the cleavage of the glycosidic bond via an oxidative mechanism using oxygen and a reductant. The full enzymatic molecular mechanisms, starting from the initial electron transfer from a reductant to oxygen activation and hydrogen peroxide formation, are not yet understood. Using quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, we have uncovered the oxygen activation mechanisms by LPMO in the presence of ascorbic acid, one of the most-used reductants in LPMOs assays. Our simulations capture the sequential formation of Cu(II)-O2 - and Cu(II)-OOH- intermediates via facile H atom abstraction from ascorbate. By investigating all the possible reaction pathways from the Cu(II)-OOH- intermediate, we ruled out Cu(II)-O• - formation via direct O-O cleavage of Cu(II)-OOH-. Meanwhile, we identified a possible pathway in which the proximal O atom of Cu(II)-OOH- abstracts a hydrogen atom from ascorbate, leading to Cu(I) and H2O2. The in-situ-generated H2O2 either converts to LPMO-Cu(II)-O• - via a homolytic reaction, or diffuses into the bulk water in an uncoupled pathway. The competition of these two pathways is strongly dependent on the binding of the carbohydrate substrate, which plays a role in barricading the in-situ-generated H2O2 molecule, preventing its diffusion from the active site into the bulk water. Based on the present results, we propose a catalytic cycle of LPMOs that is consistent with the experimental information available. In particular, it explains the enigmatic substrate dependence of the reactivity of the LPMO with H2O2.
| S-EPMC7007194 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes that today comprise a large enzyme superfamily, grouped into the distinct members AA9-AA17 (with AA12 exempted). The LPMOs have the potential to facilitate the upcycling of biomass waste products by boosting the breakdown of cellulose and other recalcitrant polysaccharides. The cellulose biopolymer is the main component of biomass waste and thus comprises a large, unexploited resource. The LPMOs work through a catalytic, oxidative reaction whose mechanism is still controversial. For instance, the nature of the intermediate performing the oxidative reaction is an open question, and the same holds for the employed co-substrate. Here we review theoretical investigations addressing these questions. The applied theoretical methods are usually based on quantum mechanics (QM), often combined with molecular mechanics (QM/MM). We discuss advantages and disadvantages of the employed theoretical methods and comment on the interplay between theoretical and experimental results.
| S-EPMC10107554 | biostudies-literature
Project description: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.
| S-EPMC6423801 | biostudies-literature
Project description:BackgroundThe discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10).ResultsHere, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation.ConclusionsTogether, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.
| S-EPMC7828015 | biostudies-literature
Project description:Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides, thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose, but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides, and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharide degradation suggest that the subtle differences in amino-acid sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO subclusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.
| S-EPMC7949027 | biostudies-literature