Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that HO, rather than O, is the preferred co-substrate of LPMOs. By controlling HO supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of HO by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled celluloseactive LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu 2+ (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms.GH61 | CBM33

SUMMARYBiomass constitutes an appealing alternative to fossil resources for the production of materials and energy. The abundance and attractiveness of vegetal biomass come along with challenges pertaining to the intricacy of its structure, evolved during billions of years to face and resist abiotic and biotic attacks. To achieve the daunting goal of plant cell wall decomposition, microorganisms have developed many (enzymatic) strategies, from which we seek inspiration to develop biotechnological processes. A major breakthrough in the field has been the discovery of enzymes today known as lytic polysaccharide monooxygenases (LPMOs), which, by catalyzing the oxidative cleavage of recalcitrant polysaccharides, allow canonical hydrolytic enzymes to depolymerize the biomass more efficiently. Very recently, it has been shown that LPMOs are not classical monooxygenases in that they can also use hydrogen peroxide (H2O2) as an oxidant. This discovery calls for a revision of our understanding of how lignocellulolytic enzymes are connected since H2O2is produced and used by several of them. The first part of this review is dedicated to the LPMO paradigm, describing knowns, unknowns, and uncertainties. We then present different lignocellulolytic redox systems, enzymatic or not, that depend on fluxes of reactive oxygen species (ROS). Based on an assessment of these putatively interconnected systems, we suggest that fine-tuning of H2O2levels and proximity between sites of H2O2production and consumption are important for fungal biomass conversion. In the last part of this review, we discuss how our evolving understanding of redox processes involved in biomass depolymerization may translate into industrial applications.

Background:The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass. Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses. Conclusion: NcLPMO9C has structural and functional features that correlate with the enzyme's catalytic capabilities. Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base.

This PDF file includes:1. Complete experimental and computational details 2. Supplementary results 3. Supplementary discussion 4. List of supplementary figures 5. Supplementary Figure 1 to 26 6. Abbreviations list 7. Supplementary references 8. QM/MM optimized xyz coordinates of states 1-9 chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) using a Dionex Bio-LC equipped with a CarboPac PA1 column as previously described. 3 To quantify A2 ox , a standard was produced in-house by treating chitobiose (Megazymes) with a chitooligosaccharide oxidase (ChitO) from Fusarium graminearum, which yields 100% conversion of chitobiose to chitobionic acid. 2,4 All chromatograms were recorded using Chromeleon 7.0 software.Chitin binding assay. The capacity of SmAA10A-WT and mutants thereof to bind β-chitin was tested by suspending 10 mg/mL of substrate in sodium phosphate buffer (50 mM, pH 7.0) in a total volume of 600 µL in 2 mL Eppendorf tubes. Reactions were started by the addition of SmAA10A (1 µM final concentration) and were incubated and stirred in an Eppendorf Comfort Thermomixer (at 40 °C, 1000 rpm). Samples were taken (100 µL) after 15, 30, 60, 120 and 240 min and immediately filtrated using a 96-well filter plate (Millipore) operated with a vacuum manifold to obtain the unbound protein fraction.In order to assess the percentage of bound proteins to the substrate, control samples with only enzyme and buffer were included, representing the maximum quantity of protein present in the samples (i.e. 100% unbound). The protein concentration in each sample was determined using the Bradford assay (Bio-Rad, Munich, Germany).H2O2 consumption experiments. H2O2 consumption by SmAA10A-WT and mutants thereof was measured according to a previously described protocol 5 using conditions that were slightly different from the standard reaction conditions described above: in order to be able to monitor the H2O2 consumption within a reasonable timescale the enzyme concentration had to be reduced and EDTA was added to reduce the background reaction of free metals-catalyzed H2O2 reduction (see Figure S6). After optimization, a standard reaction mixture contained the LPMO (50 nM) and H2O2 (100 µM) and EDTA (50 µM), without or with b-chitin (10 g.L -1 ), in sodium phosphate buffer (50 mM, pH 7.0), and the mixtures were incubated at 40 °C in a thermomixer (1000 rpm). The reactions were initiated by addition of AscA (20 µM final concentration). At regular intervals (t = 3, 6, 9, 12, 30 and 60 min), 70 µL of the reaction mixture was sampled, filtered as described above and 25 µL of the filtrate was mixed with 75 µL of a pre-mix of HRP (5 U.mL -1 final concentration) and Amplex® Red (ThermoFisher) (100 µM final concentration) in sodium phosphate buffer (50 mM pH 7.0). H2O2 concentrations waere then determined spectrophotometrically by measuring the absorbance at 540 nm in a microtiter plate reader.An H2O2 standard curve was prepared in the same conditions. Bioinformatics analysis. The sequence of the chitin-binding protein f...

The discovery of lytic polysaccharide monooxygenases (LPMOs) has revolutionized enzymatic processing of polysaccharides, in particular, recalcitrant insoluble polysaccharides, such as cellulose. These monocopper enzymes display intriguing and unprecedented catalytic chemistry, which make them highly valuable in industrial bioprocessing, but also generate considerable challenges in terms of scientific understanding and optimal implementation. One issue of particular interest is the fact that both molecular oxygen and hydrogen peroxide can drive LPMO reactions. Here, we review recent insights into the catalytic mechanism of LPMOs derived from structural, spectroscopic, and functional studies. We then turn to the question of how one can optimally harness the potential of LPMOs in biomass processing, given the current knowledge of their catalytic mechanism. Finally, we review recent, more applied studies that have addressed the importance of LPMOs in enzymatic conversion of lignocellulosic biomass and discuss how the impact of these powerful enzymes could be improved.

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from Serratia marcescens SmAA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme-substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as HO, O, and HO can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of SmAA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies.

Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.