The role of natural chlorinated hydroquinone metabolites in ligninolytic fungi

Ligninolytic Basidiomycetes have been reported to produce a wide variety of chloroaromatic compounds as secondary metabolites, which are structurally similar to environmental pollutants. Among these are chlorinated hydroquinone metabolites (CHM), such as 2-chloro-1,4-dimethoxybenzene (2Cl-14DMB), 2,6-dichloro-1,4-dimethoxybenzene (26DCl-14DMB), tetrachloro-1,4-dimethoxybenzene and tetrachloro-4-methoxyphenol, which are synthesized by 11 genera of Basidiomycetes.The biosynthesis of these chlorinated metabolites is not just a biological accident. An important physiological role of chloroaromatics, including CHM, is their antibiotic function protecting fungi from other organisms. However, since many ligninolytic white rot fungi synthesize CHM, these metabolites might also be involved in lignin degradation.The lignin degrading system of white rot fungi is composed of peroxidases, oxidases and secondary metabolites, such as veratryl alcohol (VA) and anisyl alcohol (AA). VA is an important fungal metabolite in the catalytic cycle of lignin peroxidase (LiP), one of the main ligninolytic enzymes of white rot fungi. Three functions of VA have been described: (1) VA is a redox mediator for LiP-catalyzed oxidations via intermediate formation of a cation radical (2) VA is a cofactor necessary to close the catalytic cycle of LiP (3) VA prevents the inactivation of LiP by H 2 O 2 .1,4-dimethoxybenzene (14DMB) can replace VA as a cofactor and/or redox mediator in the catalytic cycle of LiP; however, 14DMB is not a fungal metabolite. Nevertheless, 14DMB is structurally related to CHMs, which are fungal metabolites. This structural relationship between 14DMB and CHM implies that CHM may also replace VA in the catalytic cycle of LiP.The objectives of this PhD study were to determine the extent of biosynthesis of CHM among white rot fungi and to elucidate their possible physiological role in lignin degradation.For this purpose, 92 ligninolytic Basidiomycetes were screened for their ability to produce the CHMs tetrachloro-4-methoxyphenol (drosophilin A, DA) and tetrachloro-1,4-dimethoxybenzene (drosophilin A methyl ether, DAME). Five fungal strains (two strains of Bjerkandera adusta , Agaricus arvensis, Peniophora pseudopini and Phellinus fastuosus ) produced these metabolites in detectable amounts. Furthermore, the CHM production coincided with the expression of ligninolytic activity.P. fastuosus ATCC26.125 was used for further studies to elucidate if the ligninolytic enzymes from this fungus were able to use DA and DAME as substrates. The dominant ligninolytic peroxidase produced by P. fastuosus was a manganese independent peroxidase (MIP), which efficiently oxidizes phenols but not veratryl alcohol nor Mn(II). Purified MIP isozymes were able to oxidize chlorophenolic compounds, including the natural DA and the anthropogenic pentachlorophenol. MIP could not oxidize DAME or other (chlorinated) 1,4-dimethoxybenzenes.The CHMs, 2Cl-14DMB, 26DCl-14DMB, DAME and DA were examined as substrates for LiP, which is an important ligninolytic enzyme from Bjerkandera . Our results indicated that 14DMB, 2Cl-14DMB, and DA were substrates for LiP; however, 26DCl-14DMB and DAME were not. According to these results, we concluded that 26DCl-14DMB and DAME do not have a physiological role in lignin degradation, but primarily function as antibiotic agents.2Cl-14DMB, which is a LiP substrate, is synthesized by several fungi including the well studied fungus Bjerkandera sp. BOS55. We showed that 2Cl-14DMB, like 14DMB, can replace VA as a cofactor and redox mediator in the catalytic cycle of LiP.We demonstrated that LiP oxidized 2Cl-14DMB to 2-chloro-1,4-benzoquinone and two different dimers. Furthermore, EPR results showed the formation of a long-lived 2Cl-14DMB cation radical. The dimer formation and EPR results suggest that the 2Cl-14DMB cation radical is stable enough enabling it to function as a diffussible redox mediator, which can oxidize substrates at a distance of the enzyme. Our results indeed showed that 2Cl-14DMB acts as a redox mediator in the LiP-catalyzed oxidation of the polymeric dye Poly R-478, 4-methoxymandelic acid and oxalate.We examined whether 2Cl-14DMB could also replace VA as a cofactor in the LiP-catalyzed oxidation of AA. 2Cl-14DMB enormously stimulated the oxidation of AA, compared to VA. Although our results supported the theory that 2Cl-14DMB closed the catalytic cycle of LiP, we could not directly explain the high molar ratio of p-anisaldehyde formed to 2Cl-14DMB consumed. This high molar ratio indicated that the 2Cl-14DMB cation radical must be recycled back to 2Cl-14DMB. In support of this observation, AA was found to reduce the 2Cl-14DMB cation radical back to 2Cl-14DMB.Furthermore, the presence of AA to reduce the 2Cl-14DMB cation radical prevents LiP from H 2 O 2 -inactivation. According to our observations, a mechanism for the behavior of 2Cl-14DMB in the catalytic cycle of LiP and its role in AA oxidation was proposed. In the absence of AA or at low 2Cl-14DMB concentrations, a 2Cl-14DMB cation radical - LiP compound II complex is formed which ultimately results in LiP inactivation. Addition of enough reducing substrate, AA or excess 2Cl-14DMB, reduces the 2Cl-14DMB cation radical - LiP II complex releasing LiP compound II and thereby preventing the inactivation of LiP.In conclusion, CHMs were observed to be substrates for ligninolytic enzymes suggesting purposeful roles of these chlorinated metabolites. 2Cl-14DMB was identified as a stable diffussible redox mediator which could have potential application in the improvement of biobleaching and biopulping.