Laccase and Its Role in Production of Extracellular Reactive Oxygen Species during Wood Decay by the Brown Rot Basidiomycete Postia placenta

Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe³⁺, thus generating perhydroxyl radicals and Fe²⁺, which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe³⁺ tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer Postia placenta contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute P. placenta protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by P. placenta laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 10⁴ M⁻¹ s⁻¹, and measurements of the H₂O₂ produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by P. placenta laccase in wood is large enough that it likely contributes to incipient decay.Brown rot basidiomycetes are the principal recyclers of woody biomass in coniferous forest ecosystems and also the chief cause of decay in wooden structures (8, 41). Unlike the closely related white rot fungi, they degrade the cellulose and hemicellulose in wood while mineralizing little of the lignin that shields these structural polysaccharides from enzymatic attack. As a result, extensively brown-rotted wood consists primarily of an oxidized, partially cleaved residue derived from the original lignin (7, 16, 21, 22, 40). This failure to remove lignin efficiently suggests that brown rot systems contain fewer components than white rot systems and, in agreement, the recently published genome sequence of Postia placenta shows that this brown rot fungus lacks the ligninolytic peroxidases generally thought important for white rot (26). Instead, brown rot fungi appear to rely, at least during incipient decay, on small agents that can penetrate the lignin to access the polysaccharides (10, 14).A better understanding of the biodegradative agents produced by P. placenta may provide clues about what constitutes a minimally effective system for the microbial deconstruction of lignocellulose. One potential low-molecular-weight contributor is an extracellular metabolite, 2,5-dimethoxyhydroquinone (2,5-DMHQ), which has been found in cultures of P. placenta and other brown rot fungi and also shown to reduce Fe³⁺ with concomitant H₂O₂ production, thus producing hydroxyl radicals (^·OH) via the Fenton reaction (Fig. ​(Fig.1,1, reaction 6) (4, 17, 20, 29, 34, 36). Past work has shown that the chemical changes introduced by brown rot fungi into wood, cellulose, and other polymers are consistent with attack by reactive oxygen species (ROS) such as ^·OH (4, 7, 19, 21-23). A second small agent with a proposed role is extracellular oxalic acid, which P. placenta produces in sufficient quantity to acidify colonized wood to pH 2 to 4. Assays in vitro have shown that cellulose is slowly hydrolyzed at these acidities (11).Open in a separate windowFIG. 1.Chemical reactions discussed in the text. For simplicity, the HOO^·/O₂^·− acid/base pair is shown only as HOO^·. H₂Q, hydroquinone; HQ^·, semiquinone; Q, quinone.However, there is an apparent contradiction between these two mechanisms: oxalate is a strong chelator of Fe³⁺, and the resulting Fe³⁺ trioxalate complex has too negative a reduction potential to react readily with methoxyhydroquinones such as 2,5-DMHQ (28, 37). In considering this problem, we noted the surprising finding that the P. placenta genome encodes two putative laccases, enzymes that are considered atypical of brown rot fungi (26). Laccases oxidize methoxyhydroquinones to semiquinone radicals, which generally have more negative reduction potentials than their parent hydroquinones (38), and are therefore expected to be better reductants of Fe³⁺. In addition, methoxysemiquinones reduce O₂ to generate perhydroxyl radicals (HOO^·) and their conjugate base superoxide (O₂^·−), which dismutate to produce H₂O₂. Furthermore, HOO^·/O₂^·− can reduce some Fe³⁺ chelates to generate additional Fe²⁺ and can oxidize some Fe²⁺ chelates to generate additional H₂O₂ (9, 13, 33, 38). By these routes, a P. placenta laccase could bypass the requirement for the hydroquinone to react directly with Fe³⁺ and could thus generate a complete Fenton system (Fig. ​(Fig.1,1, reactions 1 to 7).Here we have expressed one of the P. placenta putative laccase genes heterologously and thus demonstrate that it encodes a typical laccase. In addition, we show that laccase activity and this particular enzyme are present in wood undergoing decay by P. placenta. Furthermore, we report that 2,5-DMHQ is present in the biodegrading wood, that it is a substrate for the P. placenta laccase, and that its oxidation during incipient wood decay requires a macromolecular component that is replaceable by P. placenta laccase. Finally, we show that the oxidation of 2,5-DMHQ by the P. placenta laccase results in significant H₂O₂ production, and we estimate that the quantity of ROS produced by this route is large enough that it probably contributes to incipient brown rot.