Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His-Gln-Tyr motif in cellulose- and chitin-oxidizing AA10B toward Arg-Glu-Phe.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface.

Ancestral sequence reconstruction as a tool to study the evolution of wood decaying fungi.

The study of evolution is limited by the techniques available to do so. Aside from the use of the fossil record, molecular phylogenetics can provide a detailed characterization of evolutionary histories using genes, genomes and proteins. However, these tools provide scarce biochemical information of the organisms and systems of interest and are therefore very limited when they come to explain protein evolution.

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in AA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity.

Reductants fuel lytic polysaccharide monooxygenase activity in a pH-dependent manner.

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H O co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.

Visible light-exposed lignin facilitates cellulose solubilization by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and are crucial for the conversion of plant biomass in Nature and in industrial applications. Sunlight promotes microbial conversion of plant litter; this effect has been attributed to photochemical degradation of lignin, a major redox-active component of secondary plant cell walls that limits enzyme access to the cell wall carbohydrates. Here, we show that exposing lignin to visible light facilitates cellulose solubilization by promoting formation of HO that fuels LPMO catalysis.

Design and biocatalytic applications of genetically fused multifunctional enzymes.

Fusion proteins, understood as those created by joining two or more genes that originally encoded independent proteins, have numerous applications in biotechnology, from analytical methods to metabolic engineering. The use of fusion enzymes in biocatalysis may be even more interesting due to the physical connection of enzymes catalyzing successive reactions into covalently linked complexes. The proximity of the active sites of two enzymes in multi-enzyme complexes can make a significant contribution to the catalytic efficiency of the reaction.

Lytic Polysaccharide Monooxygenase from with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification.

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete (TamAA9A) has been successfully expressed in and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera or have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC.

Agaricales Mushroom Lignin Peroxidase: From Structure-Function to Degradative Capabilities.

Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom .

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases.

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from sp., , and , VP from , and LiP from were produced, and their kinetic constants and reduction potentials determined.

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay.

Peroxidase evolution in white-rot fungi follows wood lignin evolution in plants.

A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme ( and ) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting ).

Different fungal peroxidases oxidize nitrophenols at a surface catalytic tryptophan.

Dye-decolorizing peroxidase (DyP) from Auricularia auricula-judae and versatile peroxidase (VP) from Pleurotus eryngii oxidize the three mononitrophenol isomers. Both enzymes have been overexpressed in Escherichia coli and in vitro activated. Despite their very different three-dimensional structures, the nitrophenol oxidation site is located at a solvent-exposed aromatic residue in both DyP (Trp377) and VP (Trp164), as revealed by liquid chromatography coupled to mass spectrometry and kinetic analyses of nitrophenol oxidation by the native enzymes and their tryptophan-less variants (the latter showing 10-60 fold lower catalytic efficiencies).

Increase of Redox Potential during the Evolution of Enzymes Degrading Recalcitrant Lignin.

To investigate how ligninolytic peroxidases acquired the uniquely high redox potential they show today, their ancestors were resurrected and characterized. Unfortunately, the transient Compounds I (CI) and II (CII) from peroxide activation of the enzyme resting state (RS) are unstable. Therefore, the reduction potentials (E°') of the three redox couples (CI/RS, CI/CII and CII/RS) were estimated (for the first time in a ligninolytic peroxidase) from equilibrium concentrations analyzed by stopped-flow UV/Vis spectroscopy.

Evolutionary convergence in lignin-degrading enzymes.

The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes.

Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date.

Background: Floudas et al. ( 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information.