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Low molecular weight chelators and phenolic compounds isolated from wood decay fungi and their role in the fungal biodegradation of wood1

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Abstract

A review of information, as well as new research results related to low molecular weight metal chelators isolated from wood decay fungi is presented. Low molecular weight compounds with metal chelating capability have been found to be produced by several white and brown rot wood decay fungi. Phenolate derivative chelators from the brown rot fungus Gloeophyllum trabeum (Gt-chelator) have been isolated by ultrafiltration (<1000 MW) and extraction with ethyl acetate. Selected chelators have been purified via HPLC and structural analysis performed by GC-MS analysis. The chelators isolated have not only a high affinity for the ferric form of iron but importantly, will mediate the reduction of this metal in redox cycling processes at pH values below neutrality. Depending on iron concentration and other environmental parameters, iron may remain bound to the chelator or can be released in the reduced ferrous form. The reduced state of iron can react with oxidants such as hydrogen peroxide where a Fenton type reaction will occur to generate oxygen radicals. The presence of the chelator in wood degraded by G. trabeum has been demonstrated by ELISA and TEM immunolabelling studies. Data are presented on the reduction of iron by Gt-chelator, the generation of oxygen radical species, and the degradation of cellulosic and phenolic compounds mediated by the Gt chelator. Also demonstrated are the `redox cycling' of the chelator as well as one-electron oxidation reactions mediated by the Gt chelator in the presence of Mn or Fe ions and hydrogen peroxide. A reaction scheme is presented and the role of oxalic acid as a phase transfer agent for iron in conjunction with low molecular weight, diffusible chelators in early through advanced stages of wood degradation is discussed. We show that oxalate plays a role in increasing the availability of iron via sequestration from iron (hydr)oxide complexes, allowing the Gt chelators, in a pH dependent reaction, to access this metal from the weak oxalate chelator. Chelators isolated from the brown rot fungus were found to reduce iron or other transition metals and sequester these metals. The chelator has a sufficiently low molecular mass to penetrate into the wood cell wall as shown in previous work. Data are presented showing that reactive oxygen species generated in the presence of the chelator can mediate the degradation of cellulosic and chlorinated phenolic compounds. We hypothesize that the Gt chelator fosters the production of reduced metals within the buffered environment of the wood cell wall and when in proximity to reactive oxygen species, such as hydrogen peroxide or other oxidants (produced in part via oxidation of the catecholate Gt chelators as well as cell wall lignocellulose components in the presence of the chelated metals), will react to form hydroxyl radicals which are capable of depolymerizing and oxidizing lignocellulose compounds. This hypothesis helps to explain the action of brown rot fungi in wood cell wall biodegradation and provides a mechanism to explain how brown rot fungi `control' the production of oxygen radicals, allowing their formation within the wood cell wall where they are effective against lignocellulose components, as opposed to being randomly generated in the extracellular environment around the fungal hyphae.

Introduction

Biodegradation of lignocellulose by fungi is of interest both because of the need for decay prevention in wood and wood products and because of the potential utility of degradative processes in biotechnological applications (Goodell, 1989; Paszczynski and Crawford, 1995). This paper serves as a review of current and past work with metal chelators isolated from white and brown rot wood degrading fungi, and details the function of `redox cycling' chelators and phenolic compounds isolated from brown rot fungi involved in wood degradation processes. The work reviewed and the data presented outline a mechanism for the production of reactive oxygen species in early through advanced stages of brown rot decay. Specifically, data are presented to support our hypotheses regarding the function of chelators from the fungus Gloeophyllum trabeum and their interaction with other fungal metabolites to produce oxygen radical species which are reactive in the chemical oxidation of both cellulosic and phenolic compounds.

Oxygen is one of the most important molecules in the life processes of aerobic organisms and is an essential component for living organisms ranging from prokaryotes to animals. However, although reactive oxygen species are known to function as intermediates in many biological reactions, they are also responsible for oxidative damage which can disrupt these systems (Cadenas, 1995; Doetsch, 1991; Halliwell and Gutteridge, 1986, Halliwell and Gutteridge, 1990). When oxygen is present in its radical forms such as superoxide and the highly reactive hydroxyl radical, it can be a potent toxin resulting in damage to genomic DNA and cellular functions (Chow, 1991). Just as oxygen radicals have been implicated as a causal agent for many diseases of human and animal life, so too have these radicals been implicated in plant and fungal metabolic disruption (Berenbaum, 1995; Dalton, 1995; Winkelmann and Winge, 1994). In studies of wood degrading fungi previous researchers have found that through enzymatic action, cation radicals are produced which are responsible for the degradation of various components of the wood cell wall (Backa et al., 1992; Barr et al., 1992; Illman et al., 1988). The pathways for this action, particularly in white rot fungi, have been reviewed by various researchers (Higuchi, 1990; Tuor et al., 1995). Ultimately though, laboratory duplication of the action of brown and white rot decay fungi via enzymatic action alone has been difficult to achieve. In part this is due to the ephemeral nature of many degradative enzymes from these organisms outside of the immediate extracellular environment of the fungal hyphae. However, other mechanistic factors in addition to enzymes also appear to be involved in wood degradation processes (Koenigs, 1974; Schmidt et al., 1981; Paszczynski et al., 1988; Daniel, 1994).

Cowling's seminal paper on the biochemistry of wood degradation (Cowling, 1961) showed that the wood cell wall was rapidly depolymerized in early stages of attack by brown rot fungi. Initial thought focused on the possibility that unidentified low molecular weight enzyme(s) could be produced by the fungi and these may be involved in the cellulose depolymerization observed. These enzymes would have to be capable of rapidly penetrating the wood cell wall to promote depolymerization. Extensive analyses of extracellular metabolites from wood degrading fungi over the last 40 years have failed to reveal any functional low molecular weight enzymes. Several research groups have studied the pore sizes of plant (Carpita et al., 1979; Tepfer and Taylor, 1981) and wood cell walls (Cowling, 1975; Reese, 1977; Stone and Scallon, 1968; Flournoy et al., 1991) and have shown that the pore size in the intact wood cell wall would not permit penetration by enzymes. In addition, immunoelectron microscopic studies have confirmed that degradative enzymes and metabolites from both white and brown rot fungi, including a variety of cellulases, ligninases, manganese peroxidases, laccase and others, are not capable of penetrating the wood cell wall in early decay stages (Kim et al., 1993Kim et al., 1991; Daniel et al., 1989Daniel et al., 1990; Srebotnik and Messner, 1991; Blanchette, 1997). To account for the unexplained depolymerization of the cell wall, various research groups have focussed on possible mechanisms employing low molecular weight agents which could potentially penetrate and initiate depolymerization of the wood cell wall. Research by Koenigs (1975)on the action of `Fenton' reagents against cellulose and wood cell wall components suggested that iron and hydrogen peroxide were involved in the production of highly reactive hydroxyl radicals which could initiate the depolymerization of cellulose in wood as:Fe2++H2O2→Fe3++HO.+HO

Hydroxyl radicals can also be generated in the presence of metals by the related Haber-Weiss reaction (Haber and Weiss, 1934) as:O2+H2O2→O2+HO+HO.

This reaction is now known to occur via the superoxide reduction of ferric iron with hydrogen peroxide oxidation of the resultant ferrous iron to produce the hydroxyl radicals shown. Further research in this area by Highley (1980)and Schmidt et al. (1981)showed that wood degraded by reactive products of the Fenton reaction displayed characteristically unique features physically and chemically similar to that of brown rotted wood, but a plausible explanation for the production and stabilization of the requisite ferrous iron in the reaction schemes proposed was not presented. Several more recent hypotheses including those involving oxalic acid have been proposed (Shimada et al., 1992; Green et al., 1991). Oxalate has been postulated to play a role in direct acid attack upon the wood cellulose and hemicellulose. It has also been suggested that oxalate may function to reduce iron III to iron II which then reacts with H2O2 to yield OḢ. However, Hyde and Wood (1995)and others (Sulzberger and Laubscher, 1995; Sedlak and Hoigné, 1993; Zepp et al., 1992) have observed that oxalate does not reduce ferric iron except as a light-dependent reaction. Therefore, oxalate cannot function as a direct catalyst of Fenton type chemical reactions in wood. Degradation of cellulose and hemicellulose in studies where photochemical effects were not controlled may have been due to photochemical reduction of iron in the presence of oxalate. In addition, in cases where samples were dried under high temperature conditions after treatment with oxalate, acid hydrolysis of cellulosic compounds would be artificially promoted. Since wood degradation normally occurs under relatively low light and temperature conditions, these factors would not be expected to play a role in natural decay processes mediated by fungi.

Other recent hypotheses concerning mechanisms related to fungal wood degradation include those involving fungal `glycopeptide' compounds (Enoki et al., 1989; Hirano et al., 1995). These compounds have been reported to bind reduced iron, as well as both reduce and oxidize the metal and are reviewed in other papers of this issue. Wood (1994)has investigated pathways for Fenton's reagent production in fungi and Hyde and Wood (1995)have also recently proposed a mechanism for brown rot degradation based on Fenton chemistry and the enzymatic generation of reduced iron species. In their model, a pH dependant autoxidation of Fe II is postulated to produce hydrogen peroxide at a distance from the hyphae to initiate Fenton reactions leading to the formation of hydroxyl radicals. In the absence of other Fenton chemistry reactants or reactive oxygen species, Fe II would not be expected to promote an oxidative degradation of cellulosic compounds found in wood (Kirk et al., 1991) and although other mechanisms for the production of hydrogen peroxide from an oxalic acid oxidase have also been suggested in brown rot fungi (Espejo and Agosin, 1991), a mechanism which would direct a site specific reaction of the free FeII and hydrogen peroxide within the wood cell wall has not been put forward. It has been established that oxidative degradation is involved in the brown rot attack of lignocellulosic materials (Kirk et al., 1989, Kirk et al., 1991) and any explanation describing wood cell wall degradation by these fungi therefore should include plausible oxidative metabolic pathways.

In this paper, data supporting the hypothesis of low molecular weight chelators functioning with other metabolites including oxalic acid in mediating the degradation of both cellulosic and phenolic compounds is presented. We propose a working model which helps to explain, at least in part, the initiation and advancement of brown rot attack in wood by the fungus Gloeophyllum trabeum. Because iron binding compounds have also been found to be produced by other decay fungi (Fekete et al., 1989) we do not rule out that this mechanism may also be employed by other brown rot fungi. Results are presented which describe mechanisms for the effective degradation of both cellulose and phenolic-type compounds by brown rot fungi. As we have not yet adequately explored the role of chelators in white rot fungi, we are not in this paper attempting to expand our hypothesis to these or other fungi.

Section snippets

Biological chelators and transition metals

To solubilize and sequester ferric iron, many organisms have evolved efficient high affinity iron aquisition systems (Neilands, 1974Neilands et al., 1987). The initial work with chelators from decay fungi presented by Fekete et al. (1989)described the screening of several white and brown rot fungi and showed that Fe III chelating compounds were produced extracellularly in fungi grown in liquid, or on solid plating medium. All fungi tested were found to produce chelating compounds. In G. trabeum

The role of iron and other metals in the fungal environment

Iron is the second most abundant metal in the lithosphere and comprises approximately 5% of the earths crust (Nealson, 1983; Budavari et al., 1989). Iron concentrations in soils can range from 1 to 20% (Hartwig and Loepper, 1993). The low solubility of many iron containing minerals, however, greatly limits its availability (Hartwig and Loepper, 1993). In the aerobic environment the most stable valence state for iron is the ferric rather than the ferrous state and although an equilibrium between

Isolation

Production of high affinity iron-binding chelators by wood decay fungi was demonstrated by Fekete et al. (1989). The presence of chelators was demonstrated by a modified chrome azural S (CAS) assay (Schwyn and Neilands, 1987), a standardized test for ferric chelation activity and was confirmed by paper electrophoresis. Ten fungi from both brown and white rot groups, including G. trabeum, were demonstrated to be CAS positive in this work.

Phenolate chelators were first isolated from G. trabeum (

Discussion

Degradation of lignin by brown rot fungi has previously been reported based on ultrastructural evidence of wood cell wall penetration and attack of lignin rich middle lamellae regions (Kim et al., 1993; Messner et al., 1985; Daniel, 1994), but in general the action of brown-rots appear to be limited to demethoxylation, demethylation and side chain cleavage of lignin derivatives (Kirk, 1975; Eriksson et al., 1990). Direct chemical evidence of lignin depolymerization by brown rot fungi is

Acknowledgements

The authors would like to acknowledge the technical assistance of B. Doyle, Y. Shao and A.-S. Hansén for portions of this research. Support from the Swedish Natural Science Research Council, the Brother Edlunds fund, and the USDA, New England Wood Utilization Research Fund was provided for portions of the research presented here. The authors would like to thank the Swedish University of Agricultural Sciences, Dept. of Forest Products, which provided facilities for the sabbatical stay of

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