Search

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Significance Lytic polysaccharide monooxygenases (LPMOs) have unique catalytic centers, at which a single copper catalyzes the oxidative cleavage of a glycosidic bond. The mechanism by which LPMOs activate molecular oxygen is key to understanding copper (bio)catalysis but remains poorly understood, largely because the insoluble and heterogeneous nature of LPMO substrates precludes the use of usual laboratory techniques. Using an integrated NMR/EPR approach, we have unraveled structural and electronic details of the interactions of an LPMO from Bacillus licheniformis and β-chitin. EPR spectroscopy on uniformly isotope 15N-labeled 63Cu(II)-LPMO provided insight into substrate-driven rearrangement of the copper coordination sphere that predisposes the enzyme for O2 activation., Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x2−y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Probing the detailed interaction between lytic polysaccharide monooxygenases (LPMOs) and their polysaccharide substrates is key to revealing further insights into the mechanism of action of this class of enzymes on recalcitrant biomass. This investigation is somewhat hindered, however, by the insoluble nature of the substrates, which precludes the use of most optical spectroscopic techniques. Herein, we report a new semi-oriented EPR method which evaluates directly the binding of cellulose-active LPMOs to crystalline cellulose. We make use of the intrinsic order of cellulose fibres in Apium graveolens (celery) to orient the LPMO with respect to the magnetic field of an EPR spectrometer. The subsequent angle-dependent changes observed in the EPR spectra can then be related to the orientation of the g matrix principal directions with respect to the magnetic field of the spectrometer and, hence, to the binding of the enzyme onto the cellulose fibres. This method, which does not require specific modification of standard CW-EPR equipment, can be used as a general procedure to investigate LPMO-cellulose interactions.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite

Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen...

Background The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate. Results Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases. Conclusion Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

International audience; Alternatives to petroleum-based chemicals are highly sought-after for ongoing efforts to reduce the damaging effects of human activity on the environment. Copper radical oxidases from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts because they oxidize primary alcohols in a chemoselective manner without complex organic cofactors. However, despite numerous studies on canonical galactose oxidases (GalOx, EC 1.1.3.9) and engineered variants, and the recent discovery of a Colletotrichum graminicola copper radical alcohol oxidase (AlcOx, EC 1.1.3.13), the catalytic potentials of very few AA5_2 members have been characterized. Guided by the sequence similarity network and phylogenetic analyses, we targeted a distinct paralog from the fungus C. graminicola as a representative member of a large uncharacterized subgroup of AA5_2. Through recombinant production and detailed kinetic analysis, we demonstrated that this enzyme is weakly active toward carbohydrates but efficiently catalyzes the oxidation of aryl alcohols to the corresponding aldehydes. As such, this represents the initial characterization of a demonstrable aryl alcohol oxidase (AAO, EC 1.1.3.7) in AA5, an activity which is classically associated with flavin-dependent glucose-methanol-choline (GMC) oxidoreductases of Auxiliary Activity Family 3 (AA3). X-ray crystallography revealed a distinct multidomain architecture comprising an N-terminal PAN domain abutting a canonical AA5 seven-bladed propeller catalytic domain. Of direct relevance to biomass processing, the wild-type enzyme exhibits the highest activity on the primary alcohol of 5-hydroxymethylfurfural (HMF), a product of significant interest in the lignocellulosic biorefinery concept. Thus, the chemoselective oxidation of HMF to 2,5-diformylfuran (DFF) by C. graminicola aryl alcohol oxidase (CgrAAO) from AA5 provides a fundamental building block for chemistry via biotechnology.

Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV–vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)–tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)–tyrosyl radical center entails the formation of significant Cu(II)–(●OTyr) overlap, which in turn requires that the plane of the d(x2–y2) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a “hole-hopping” charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.

Lytic polysaccharide monooxygenases (LPMOs, also known as PMOs) are a recently discovered family of enzymes that play a key role in the breakdown of polysaccharide substrates. The ability of LPMOs to introduce chain breaks, using an oxidative mechanism, has particularly attracted attention as the world seeks more cost-effective and environmentally friendly ways of producing second-generation biofuels for the future. LPMOs are copper-dependent enzymes and have an unusual active site which includes the N-terminal residue of the protein in the copper coordination sphere. This N-terminal histidine side chain is also methylated in fungal enzymes, the molecular reason for which is still a debated topic. The production of these enzymes poses several challenges if we are to understand their chemical mechanisms. Here, we describe the methods that have been used in the field to produce LPMOs and provide information on the workflows that we use for our electron paramagnetic resonance (EPR) spectroscopy experiments. EPR has been a particularly powerful tool in the study of these enzymes and our objective with this chapter is to provide some helpful information for researchers for whom this technique might be daunting or theoretically difficult to access.

Lytic polysaccharide monooxygenases (LPMOs, also known as PMOs) are a recently discovered family of enzymes that play a key role in the breakdown of polysaccharide substrates. The ability of LPMOs to introduce chain breaks, using an oxidative mechanism, has particularly attracted attention as the world seeks more cost-effective and environmentally friendly ways of producing second-generation biofuels for the future. LPMOs are copper-dependent enzymes and have an unusual active site which includes the N-terminal residue of the protein in the copper coordination sphere. This N-terminal histidine side chain is also methylated in fungal enzymes, the molecular reason for which is still a debated topic. The production of these enzymes poses several challenges if we are to understand their chemical mechanisms. Here, we describe the methods that have been used in the field to produce LPMOs and provide information on the workflows that we use for our electron paramagnetic resonance (EPR) spectroscopy experiments. EPR has been a particularly powerful tool in the study of these enzymes and our objective with this chapter is to provide some helpful information for researchers for whom this technique might be daunting or theoretically difficult to access.

A structural unit found in the active site of some copper proteins, the histidine brace, is comprised of an N-terminal histidine that chelates a single copper ion through its amino terminus NH2 and the π–N of its imidazole side chain. Coordination is completed by the τ-N of a further histidine side chain, to give an overall N3 T-shaped coordination at the copper ion. The histidine brace appears in several proteins, including lytic polysaccharide monooxygenases LPMOs and particulate methane monooxygenases pMMOs, both of which catalyse the oxidation of substrates with strong C–H bonds (bond dissociation enthalpies ~100 kcal mol–1). As such, the copper histidine brace is the focus of research aimed at understanding how nature catalyses the oxidation of unactivated C–H bonds. In this Perspective, we evaluate these studies, which further give bioinspired direction to coordination chemists in the design and preparation of small molecule copper oxidation catalysts. The histidine brace found in certain copper oxidases enables the oxidation of strong C–H bonds in organic substrates. This Perspective highlights and discusses the possible structural and electronic features of this motif and how these features underlie its role in challenging oxidative catalysis.

International audience; Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.

The coordination modes of the [Au(PPh3)](+) cation to metal alkynyl complexes have been investigated. On addition to ruthenium, a vinylidene complex, [Ru(η(5)-C5H5)(PPh3)2([double bond, length as m-dash]C[double bond, length as m-dash]CPh{AuPPh3})](+), is obtained while addition to a gold(iii) compound gives di- and trinuclear gold complexes depending on the conditions employed. In the trinuclear species, a gold(i) cation is sandwiched between two gold(iii) alkynyl complexes, suggesting that coordination of multiple C-C triple bonds to gold is facile.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which 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 the first 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.

Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications., Auxilliary Activity Family 5 (AA5) comprises mononuclear copper radical oxidases with catalytic diversity that is not well characterised. Here, structural, phylogenetic and biochemical analyses advance our understanding of the potential biological and biotechnology functions of these proteins.