Abstract
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.
Highlights
Plant biomass is an abundant renewable resource that has attracted an increasing interest during the last decades in the context of lignocellulose biorefinery [1,2]
Antioxidants 2021, 10, 1446 are the only organisms able to extensively mineralize native lignin [9,14,15]. They use an array of oxidative enzymatic tools for its extracellular degradation, among which the ligninolytic peroxidases play a major role [9,16]. These enzymes are part of class-II of the peroxidase–catalase superfamily [17] and, according to the oxidation sites in their molecular architecture, are classified into three families [16]: (i) manganese peroxidases (MnPs) harboring a Mn-binding site where Mn2+ is oxidized to Mn3+, which acts as a diffusible mediator both oxidizing the minor phenolic moiety of lignin and generating strong oxidizers by initiating lipid peroxidation [18]; (ii) lignin peroxidases (LiPs) containing a solvent-exposed catalytic tryptophan where they directly oxidize high redox-potential non-phenolic aromatic substrates [20,21]; and (iii) versatile peroxidases (VPs) accommodating in a single enzyme the catalytic sites previously described for MnPs and LiPs [22]
16 August 2021), a fungus belonging to the order Agaricales, contains a gene encoding a putative LiP enzyme (JGI protein ID# 705809, identified by the presence of a characteristic solvent exposed tryptophan residue and the absence of a Mn2+ -oxidation site)
Summary
Plant biomass is an abundant renewable resource that has attracted an increasing interest during the last decades in the context of lignocellulose biorefinery [1,2]. Antioxidants 2021, 10, 1446 are the only organisms able to extensively mineralize native lignin [9,14,15] They use an array of oxidative enzymatic tools for its extracellular degradation, among which the ligninolytic peroxidases play a major role [9,16]. These enzymes are part of class-II of the peroxidase–catalase superfamily [17] and, according to the oxidation sites in their molecular architecture, are classified into three families [16]: (i) manganese peroxidases (MnPs) harboring a Mn-binding site where Mn2+ is oxidized to Mn3+ , which acts as a diffusible mediator both oxidizing the minor phenolic moiety of lignin and generating strong oxidizers by initiating lipid peroxidation [18] (members of the short MnP subfamily are able to oxidize phenolic compounds in direct contact with the heme cofactor [19]);
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