Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague.
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ABSTRACT:
SUBMITTER: Sabbadin F
PROVIDER: S-EPMC8393829 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member (CflaLPMO10D) as a chitin-active LPMO. Both the full-length CflaLPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that CflaLPMO10D is strictly active on the β-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on β-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.
| S-EPMC9361826 | biostudies-literature
Project description:Pseudomonas aeruginosa secretes diverse proteins via its type 2 secretion system, including a 39 kDa chitin-binding protein, CbpD. CbpD has recently been shown to be a lytic polysaccharide monooxygenase active on chitin and to contribute substantially to virulence. To date, no structure of this virulence factor has been reported. Its first two domains are homologous to those found in the crystal structure of Vibrio cholerae GbpA, while the third domain is homologous to the NMR structure of the CBM73 domain of Cellvibrio japonicus CjLPMO10A. Here, the 3.0 Å resolution crystal structure of CbpD solved by molecular replacement is reported, which required ab initio models of each CbpD domain generated by the artificial intelligence deep-learning structure-prediction algorithm RoseTTAFold. The structure of CbpD confirms some previously reported substrate-specificity motifs among LPMOAA10s, while challenging the predictive power of others. Additionally, the structure of CbpD shows that post-translational modifications occur on the chitin-binding surface. Moreover, the structure raises interesting possibilities about how type 2 secretion-system substrates may interact with the secretion machinery and demonstrates the utility of new artificial intelligence protein structure-prediction algorithms in making challenging structural targets tractable.
| S-EPMC9344471 | biostudies-literature
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:Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a β-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.
| S-EPMC5778151 | biostudies-literature
Project description: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 of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
| S-EPMC4817220 | 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 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