Structure-function characterization of two enzymes from novel subfamilies of manganese peroxidases secreted by the lignocellulose-degrading Agaricales fungi Agrocybe pediades and Cyathus striatus.

BACKGROUND: Manganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure-function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule. RESULTS: Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD-referred to as Ape-MnP1 and Cst-MnP1, respectively-were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation. CONCLUSIONS: Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure-function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.

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 Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.

Optimizing operational parameters for the enzymatic production of furandicarboxylic acid building block.

BACKGROUND: 2,5-Furandicarboxylic acid (FDCA) is a precursor for green plastics due to its structural similarity to terephthalic acid, a common precursor of oil-derived polymers, and its potential production from sugars obtained from plant biomass. Hydroxymethylfurfural oxidase (HMFO) has been reported as a promising biocatalyst for FDCA production since it can convert bio-based 5-hydroxymethylfurfural (HMF) into FDCA building block. This three-step oxidation reaction occurs through the diformylfuran and 2,5-formylfurancarboxylic acid (FFCA) intermediates. Several efforts have been made for the development of HMFO variants that increase FDCA yields by improving their activities over the reaction intermediates. However, there is still limited insight into how operational conditions can influence these enzymatic reactions. The setup of optimal reaction conditions would enable to understand potential problems hampering the effective industrial production of this bioplastic precursor using HMFO as biocatalyst. RESULTS: In this work, several parameters affecting the performance of Methylovorus sp HMFO oxidizing HMF have been analyzed for the wild-type enzyme, and its V367R and W466F single variants, V367R/W466F double variant, and I73V/H74Y/G356H/V367R/T414K/A419Y/A435E/W466F (8BxHMFO) octuple variant. Our results show how the oxidation of HMF by HMFO enzymes is highly influenced by pH, with different optimal pH values for the different improved variants. Moreover, the enzymes are not stable at high hydrogen peroxide concentrations and their activity is inhibited by the FFCA intermediate in a pH-dependent way. These limitations can be efficiently overcome with the addition of catalase to the reaction medium, which removes the hydrogen peroxide formed during the oxidations, and the controlled dosage of the substrate to limit the amount of FFCA accumulated in the reaction. The different behavior of wild-type HMFO and its variants against pH, hydrogen peroxide and FFCA highlights the importance of considering each variant as an individual enzyme with its own operational conditions for an eventual industrial FDCA production. CONCLUSIONS: This work provides information of those parameters that condition a high production of FDCA by HMFO. Unraveling these factors allowed to increase the FDCA yields by using the most stable enzymes at their optimal pH for HMF oxidation, removing the peroxide with catalase, and avoiding FFCA accumulation by controlling substrate and/or enzyme concentration. These above findings will be useful when planning a future scale-up of these conversions and will provide new viewpoints for the design of HMFO variants that render a more effective performance during HMF conversion into FDCA.

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat-straw transformation. Up-regulated and constitutive glycoside-hydrolases, polysaccharide-lyases, and carbohydrate-esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl-alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.