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Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

ABSTRACT: Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA+•), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetramethoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA+•, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA+• and that a different mechanism produces small ligninolytic oxidants during white rot.

SUBMITTER: Houtman CJ

PROVIDER: S-EPMC5880153 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Houtman Carl J CJ Maligaspe Eranda E Hunt Christopher G CG Fernández-Fueyo Elena E Martínez Angel T AT Hammel Kenneth E KE

The Journal of biological chemistry 20180209 13

Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA<sup>+•</sup>), releasi ...[more]

PMID: 29462790

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Project description:BACKGROUND:Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities. RESULTS:In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer. CONCLUSIONS:A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.

Project description:BackgroundThe genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation.ResultsHeterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences.ConclusionsThe analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing enzymes of biotechnological interest.

Dataset Information

Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Publications

Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Similar Datasets

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