Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

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Abstract

Lytic polysaccharide monooxygenases have attracted vast attention due to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II)–dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex. <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/7d3c2a40-174e-4bdb-81eb-d7952a6b15cf/PubMedCentral/image/nihms851043u1.jpg"/> </div> X-ray crystallography produces the first structure of an LPMO enzyme with an activated dioxo species bound in the plane of the histidine brace. A “pre-binding” site for molecular oxygen is also identified and characterized with neutron protein crystallography and DFT calculations.

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Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

Anthony Levasseur, Elodie Drula, Vincent Lombard … (2013)

Background Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Results Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). Conclusions The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.

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An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.

Gustav Vaaje-Kolstad, Bjørge Westereng, Svein J. Horn … (2010)

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 H(2)(18)O or (18)O(2). 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.

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Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

Paul Harris, Ditte Welner, K. C. McFarland … (2010)

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.