PTMs of the lytic polysaccharide monooxygenase of Pseudomonas aeruginosa
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Pseudomonas Aeruginosa
SUBMITTER: 
Magnus Arntzen
LAB HEAD: Gustav Vaaje-Kolstad
PROVIDER: PXD018769 | Pride | 2021-01-12
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 191001_OG1.mgf | Mgf | |||
| 191001_OG1.raw | Raw | |||
| 191001_OG1_2.mgf | Mgf | |||
| 191001_OG1_2.raw | Raw | |||
| 191001_OG2.mgf | Mgf |
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Project description:Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered enzyme family that cleave polysaccharides by oxidation. Despite proposed roles in bacterial virulence, no direct functional data exist to validate these claims. Here we show that CpbD, the LPMO of the opportunistic pathogen Pseudomonas aeruginosa (PA) is a virulence factor that promotes survival of the bacterium in whole human blood. CbpD was also shown to cleave the glycosidic bonds of the model substrate by an oxidative reaction, a feature that promoted by azurin and pyocyanin. Two redox-active virulence factors co-secreted with the LPMO. Combination of homology modelling, molecular dynamics simulations and small angle X-ray scattering demonstrated that CbpD is a monomeric tri-modular enzyme with highly flexible domain linkers, where domain positioning may be influenced by post translational modifications. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection and enhanced bacterial clearance in vivo. Improved bacterial survival of the wildtype was not attributable to dampening of pro-inflammatory responses by the protein, either ex vivo or in vivo. Further quantification of complement products in whole human blood revealed that CbpD reduced the performance of the terminal complement cascade. Importantly, CbpD inactivation by active site mutations abolished both enzyme activity in vitro and function ex vivo, indicating that ligand oxidation is crucial for CbpD virulence function. Finally, profiling of the bacterial and splenic proteome showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial defense systems in vitro and the targeting of distinct host proteins/pathways in vivo.
2021-01-28 | PXD021888 | Pride
Project description:Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered enzyme family that cleave polysaccharides by oxidation. Despite proposed roles in bacterial virulence, no direct functional data exist to validate these claims. Here we show that CpbD, the LPMO of the opportunistic pathogen Pseudomonas aeruginosa (PA) is a virulence factor that promotes survival of the bacterium in whole human blood. CbpD was also shown to cleave the glycosidic bonds of the model substrate by an oxidative reaction, a feature that promoted by azurin and pyocyanin. Two redox-active virulence factors co-secreted with the LPMO. Combination of homology modelling, molecular dynamics simulations and small angle X-ray scattering demonstrated that CbpD is a monomeric tri-modular enzyme with highly flexible domain linkers, where domain positioning may be influenced by post translational modifications. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection and enhanced bacterial clearance in vivo. Improved bacterial survival of the wildtype was not attributable to dampening of pro-inflammatory responses by the protein, either ex vivo or in vivo. Further quantification of complement products in whole human blood revealed that CbpD reduced the performance of the terminal complement cascade. Importantly, CbpD inactivation by active site mutations abolished both enzyme activity in vitro and function ex vivo, indicating that ligand oxidation is crucial for CbpD virulence function. Finally, profiling of the bacterial and splenic proteome showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial defense systems in vitro and the targeting of distinct host proteins/pathways in vivo.
2021-01-28 | PXD017971 | Pride
Project description:Bacterial lytic polysaccharide monooxygenases (LPMOs) are understudied in the context of infection disease. We demonstrated that Pseudomonas aeruginosa LPMO CbpD is a virulence determinant in pneumonia. Moreover, Mice immunized with CbpD generate robust antibody responses that provide protection against lethal lung infection
2023-07-26 | PXD039298 | Pride
Project description:Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in biotechnological purposes. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identified the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirmed that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies showed endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs resulted in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this unique post-translational modification.
2023-07-14 | PXD037734 | Pride
Project description:BackgroundThe availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media.ResultsAn LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction.ConclusionsThe biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.
| S-EPMC6894463 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurospora crassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
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Project description:Bacterial lytic polysaccharide monooxygenases (LPMO10s) use redox chemistry to cleave glycosidic bonds in the two foremost recalcitrant polysaccharides found in nature, namely cellulose and chitin. Analysis of correlated mutations revealed that the substrate-binding and copper-containing surface of LPMO10s composes a network of co-evolved residues and interactions, whose roles in LPMO functionality are unclear. Here, we mutated a subset of these correlated residues in a newly characterized C1/C4-oxidizing LPMO10 from Micromonospora aurantiaca (MaLPMO10B) to the corresponding residues in strictly C1-oxidizing LPMO10s. We found that surface properties near the catalytic copper, i.e. side chains likely to be involved in substrate positioning, are major determinants of the C1:C4 ratio. Several MaLPMO10B mutants almost completely lost C4-oxidizing activity while maintaining C1-oxidizing activity. These mutants also lost chitin-oxidizing activity, which is typically observed for C1/C4-oxidizing, but not for C1-oxidizing, cellulose-active LPMO10s. Selective loss in C1-oxidizing activity was not observed. Additional mutational experiments disclosed that neither truncation of the MaLPMO10B family 2 carbohydrate-binding module nor mutations altering access to the solvent-exposed axial copper coordination site significantly change the C1:C4 ratio. Importantly, several of the mutations that altered interactions with the substrate exhibited reduced stability. This effect could be explained by productive substrate binding that protects LPMOs from oxidative self-inactivation. We discuss these stability issues in view of recent findings on LPMO catalysis, such as the involvement of H2O2 Our results show that residues on the substrate-binding surface of LPMOs have co-evolved to optimize several of the interconnected properties: substrate binding and specificity, oxidative regioselectivity, catalytic efficiency, and stability.
| S-EPMC5787815 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In this work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods. We used HADDOCK to perform protein-protein docking experiments on all 20 combinations and subsequently to select four complexes for extensive molecular dynamics simulations. The potential of mean force is computed for a rotation of the cytochrome domain relative to LPMO. We find that the LPMO loops are largely responsible for the preferred orientations of the cytochrome domains. This leads us to postulate a hybrid version of NcLPMO9F, with exchanged loops and predicted altered cytochrome binding preferences for this variant. Our work provides insight into the possible mechanisms of electron transfer between the two protein systems, in agreement with and complementary to previously published experimental data.
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Project description:The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2 O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra- and intermolecular electron transfer rates of the engineered CDH were similar to the wild-type CDH, meaning that the mutations had not compromised CDH's role as an electron donor. These results support the notion of H2 O2 -driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH-generated H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.
| S-EPMC7078924 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein-carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.
| S-EPMC5651836 | biostudies-literature