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.
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.
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 copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular LPMO10C from We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length LPMO10C resides in the CBM. Comparison of the catalytic performance of full-lengthLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general.
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