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Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H 2 O 2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with k cat / K mH2O2 values in the order of 10 5 –10 6  M −1 s −1 . Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases. Lytic polysaccharide monooxygenases (LPMOs) catalyze the hydroxylation of glycosidic bonds in polysaccharides, but the catalytic properties of these monocopper enzymes remain poorly characterized. Here authors employ competition between reference enzymes and LPMOs for the H 2 O 2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation.

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H₂¹⁸O or ¹⁸O₂. There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of recalcitrant substrates such as chitin or cellulose. Recent work has shown that LPMOs catalyze fast peroxygenase reactions and that, under commonly used reaction set-ups, access to in situ generated H 2 O 2 likely limits catalysis. Based on a hypothesis that the impact of a cellulose-binding module (CBM) on LPMO activity could relate to changes in in situ H 2 O 2 production, we have assessed the interplay between CBM-containing Sc LPMO10C and its truncated form comprising the catalytic domain only ( Sc LPMO10C TR ). The results show that truncation of the linker and CBM leads to elevated H 2 O 2 production and decreased enzyme stability. Most interestingly, combining the two enzyme forms yields strong synergistic effects, which are due to the combination of high H 2 O 2 generation by Sc LPMO10C TR and efficient productive use of H 2 O 2 by the full-length enzyme. Thus, cellulose degradation becomes faster, while enzyme inactivation due to off-pathway reactions with excess H 2 O 2 is reduced. These results underpin the complexity of ascorbic acid-driven LPMO reactions and reveal a potential mechanism for how LPMOs may interact synergistically during cellulose degradation.

Lytic polysaccharide (mono)oxygenases (LPMOs) perform oxidative cleavage of polysaccharides, and are key enzymes in biomass processing and the global carbon cycle. It has been shown that LPMO reactions may be driven by light, using photosynthetic pigments or photocatalysts, but the mechanism behind this highly attractive catalytic route remains unknown. Here, prompted by the discovery that LPMOs catalyze a peroxygenase reaction more efficiently than a monooxygenase reaction, we revisit these light-driven systems, using an LPMO from Streptomyces coelicolor ( Sc AA10C) as model cellulolytic enzyme. By using coupled enzymatic assays, we show that H 2 O 2 is produced and necessary for efficient light-driven activity of Sc AA10C. Importantly, this activity is achieved without addition of reducing agents and proportional to the light intensity. Overall, the results highlight the importance of controlling fluxes of reactive oxygen species in LPMO reactions and demonstrate the feasibility of light-driven, tunable enzymatic peroxygenation to degrade recalcitrant polysaccharides. Lytic polysaccharide (mono)oxygenases (LPMOs) perform oxidative cleavage of polysaccharides. Here, the authors showed that the light-driven activity of LPMOs is dependent on hydrogen peroxide availability and can be controlled via the light intensity provided.

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. Here, we attempt to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called “Ceres”) or a bacterial catalase-peroxidase from Klebsiella sp ., respectively. Reproducing previously described experiments, we do not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted. Recently, two oxidative enzymes have been reported to degrade extremely recalcitrant plastics, PVC and PE. Here, the authors show that these encouraging previous results could not be reproduced, and provide possible reasons for why the data may have been misinterpreted.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and are crucial for the conversion of plant biomass in Nature and in industrial applications. Sunlight promotes microbial conversion of plant litter; this effect has been attributed to photochemical degradation of lignin, a major redox-active component of secondary plant cell walls that limits enzyme access to the cell wall carbohydrates. Here, we show that exposing lignin to visible light facilitates cellulose solubilization by promoting formation of H 2 O 2 that fuels LPMO catalysis. Light-driven H 2 O 2 formation is accompanied by oxidation of ring-conjugated olefins in the lignin, while LPMO-catalyzed oxidation of phenolic hydroxyls leads to the required priming reduction of the enzyme. The discovery that light-driven abiotic reactions in Nature can fuel H 2 O 2 -dependent redox enzymes involved in deconstructing lignocellulose may offer opportunities for bioprocessing and provides an enzymatic explanation for the known effect of visible light on biomass conversion. Degradation of plant biomass, comprised of cellulose and polyaromatic lignin, is promoted by light. Here, the authors show that light promotes lignin-catalyzed generation of hydrogen peroxide, which is used by redox enzymes to degrade cellulose.

Background Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efficiencies of various LPMO-reductant combinations remains largely unexplained. Results In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomycete, which we have used to look more closely at the effects of the reductant and copper ions on the LPMO reaction. Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of H2O2 resulting from oxidation of the reductant. Conclusion The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H2O2.

Chitin, a polymer of β(1-4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.

Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu ²⁺ (K d = 55 nM; from isothermal titration calorimetry) and higher preference for Cu ¹⁺ (K d ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu ²⁺ of 275 mV using a thermodynamic cycle). Strong binding of Cu ¹⁺ was also reflected in a reduction in the p K ₐ values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a ²H/ ¹H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa , Nc LPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by Nc LPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that Nc LPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.