Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

Formation of a Copper(II)-Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2.

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.

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that 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 a 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.

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Multicomponent carbohydrase system from Trichoderma reesei: A toolbox to address complexity of cell walls of plant substrates in animal feed.

A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and ß-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different ß-glucanases (both endo-ß-1,3(4)-glucanase and endo-ß-1,3-glucanase), cellulases and a ß-glucosidase in the T. reesei fermentation effectively reduced polymerization of both ß-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific ß-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.

Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose.

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.

Learning from oligosaccharide soaks of crystals of an AA13 lytic polysaccharide monooxygenase: crystal packing, ligand binding and active-site disorder.

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch-derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO-substrate complex was reported, giving insights into the interaction of LPMOs with ß-linked substrates (Frandsen et al., 2016). The LPMOs acting on α-linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on ß-linked glycosidic bonds (families AA9-AA11), as revealed from the first determined structure (Lo Leggio et al., 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme, Aspergillus oryzae AA13, are presented. Crystals obtained in the presence of high zinc-ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper-containing structure. One structure with an ordered zinc-bound active site was solved at 1.65 Šresolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. Despite similar unit-cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate-binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His-ligand conformation that is incompatible with metal-ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive.

Oxidoreductases on their way to industrial biotransformations.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by ß-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.