Chapter 3
Free Radical Reactions of Wood-Degrading Fungi N. Scott R e a d i n g , Kevin D. W e l c h , a n d Steven D. A u s t *
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Chemistry and Biochemistry Department, Biotechnology Center, Utah State University, Logan, U T 84322-4705 Corresponding author: email:
[email protected] *
Wood-rotting fungi catalyze, through a unique system of enzymes and chemicals, the degradation and mineralization of the structural polymers in wood. The most complex of these polymers is lignin, which is a heterogeneous, stereo-irregular, three-dimensional polymer that is very resistant to enzymatic degradation due to the lack of repeating and readily hydrolyzable bonds. The biodegradation systems of the wood -rotting fungi are based largely on free-radical reactions catalyzed by a variety of extracellular enzymes. The wood -degrading systems employed by these fungi can catalyze both direct and indirect (or mediated) oxidative and reductive reactions. Fungi are able to catalyze other reactions that support the free-radical nature of these biodegradative systems and together favor depolymerization and degradation rather than synthetic or polymerization reactions.
Of the more than a million species of fungi, only a relative few, specialized and ubiquitous fungi, belonging to the phyla Basidiomycota and Ascomycota have the ability to degrade wood (1). These wood-decaying fungi are frequently grouped according to the characteristics of the wood during degradation, which reflects fundamental differences in their enzymatic and non-enzymatic activities. These are the white-rot, brown-rot, and soft-rot fungi. The white-rot fungi comprise the largest group of wood-rot fungi, having the capability to degrade
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© 2003 American Chemical Society Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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17 and mineralize all the major components of wood (cellulose, hemicellulose, and lignin), resulting in wood that becomes progressively more fragile as it decays. The white color associated with these fungi is a result of the rapid degradation of lignin, which exposes the more slowly degraded cellulose that is white in appearance. Brown-rot fungi comprise approximately 10% of all wood decay fUngi and primarily attack conifers. Most of these fungi are found in the class Polyporaceae. Brown-rot fungi primarily degrade cellulose aiid hemicellulose, leaving partially oxidized, brown lignin, which results in weakened wood structure such that the wood breaks up into cubic particles that comprise a major component of humus. Soft-rot fimgi are the least specialized of the wood-rot fungi, often requiring significant levels of nitrogen from the surrounding environment to effectively decay wood. Soft-rot fungi typically occur in wood with high water content and degrade primarily cellulose and hemicellulose. These fungi primarily belong to the phyla Ascomycota and are common decomposers of cellulose in soil.
WOOD STRUCTURE AND FREE-RADICALS Wood-rotting fungi need to be specially adapted to overcome three major defense strategies in order to degrade wood. Two of these defense strategies have a biological basis, while the third is chemically based. The biological defenses against wood degradation are nutrient availability and the presence of compounds toxic to fungi. Wood typically has a very low content of nitrogen and phosphorus (2), two elements that are important for microbial growth. The average nitrogen content for hardwoods and softwoods is 0.09% of the dry weight of wood with an average carbon to nitrogen ratio of 600:1 (2). Woodrotting fungi have adapted to this constraint by using nutrient limitation as a key factor involved in expression of their wood-degrading systems (3). The presence of potentially toxic chemicals within non-living heartwood, such as tannins in deciduous trees and a variety of phenolic compounds in coniferous trees, prevent or limit wood-rot fungi from colonizing living trees (7). The third defense strategy used by trees to decrease their susceptibility to degradation is the formation of complex organic compounds that limit the availability of easily usable substrates, such as simple sugars and starches. The principle components of wood are cellulose, hemicellulose and lignin. Cellulose comprises 40-50% (dry weight) of wood and is composed of long, linear chains of (3-1,4-linked glucose (7). Cellulose polymers hydrogen bond to form fibrils. Hemicellulose comprises 25-40% (dry weight) of wood and is a complex combination of relatively short polymers of xylose, arabinose, galactose, mannose, and glucose (7) with acetyl and uronic extensions. Hemicellulose
Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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18 hydrogen bonds to cellulose fibrils and also covalently links with lignin, creating a complex web of bonds that provide structural strength to wood. Lignin comprises 20-35% (dry weight) of wood and is a complex, stereoirregular, three-dimensional polymer composed of /?-hydroxycinnamyl alcohols, most notably £>-coumaryl, coniferyl, and synapyl alcohols, which are cross-linked to each other with a variety of chemical bonds (7). Lignin forms an amorphic complex with hemicellulose to encapsulate cellulose, thereby reducing the bioavailability of these two cell wall constituents. The lack of readily hydrolyzable, repeating linkages makes lignin particularly difficult to biodegrade and thus acts as an effective barrier against microbial attack. The biosynthesis of lignin in plants is believed to occur by a peroxidasecatalyzed, free-radical polymerization of the ^-hydroxycinnamyl alcohols. The relative concentration of each phenylpropanoid precursor, which differs with each plant species and particular cell types, determines the final structure of lignin. Peroxidases oxidize the phenylpropanoid compounds to generate radicals that combine randomly with other lignin precursors and lignin substructures to form a chemically unique, three-dimensional polymer linked through a variety of carbon-carbon and carbon-oxygen bonds. The number and variation of chemically unique structures that comprise lignin make its degradation a complicated process that requires a nonhydrolytic system that is extracellular, and nonspecific. The lignin degradation system developed by wood-degrading fungi (in particular white-rot fungi) is based largely on free-radical reactions catalyzed by a relatively small number of enzymes. Thus, the fungal, freeradical based, enzymatic system, in conjunction with other extracellular fungal activities, is able to degrade the large number of diverse chemical structures that are found in wood. The process of lignin synthesis results in a highly oxidized polymer. Therefore, to effectively degrade and mineralize lignin reductive as well as oxidative reactions are required, both of which must occur aerobically. These reactions must be balanced or otherwise controlled to prevent redox cycling and free-radical based polymerization of the degradation products. Additionally, the oxidizing and reducing equivalents need to be unique and continuously produced since extracellular regeneration would be improbable. This precludes the use of common biological compounds for reducing or oxidizing equivalents, such as N A D P H , which would be difficult to regenerate once released extracellularly. A n advantage of the extracellular formation of the free-radical species is their ability to diffuse away from their site of origin and mediate indirect reactions with the insoluble lignin polymer. Thus, the smaller, diffusible radicals achieve a greater area of reactivity than could be achieved by reactions catalyzed by enzymes or the fungi directly. This also causes free-radical reactions to occur away from the fungal mycellium preventing self-inflicted damage to the fungus.
Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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INITIATION OF FREE-RADICAL REACTIONS The extracellular enzymes of wood-degrading fungi involved in the production of free-radicals include lignin peroxidases (LiP), manganesedependent peroxidases (MnP), cellobiose dehydrogenase (CDH) and laccases. Other factors related to free-radical generation by these enzymes are hydrogen peroxide ( H 0 ) , oxalate, small molecule mediators, methyl transferases, and the plasma membrane redox potential. The free-radical reactions generated by these systems lead to both direct and indirect (mediated) oxidations and reductions. The ligninolytic peroxidases have been isolated from white-rot fungi, laccases have been isolated from the white-rot and brown-rot fungi, and C D H has been isolated from species from each group of wood-rotting fungi (4). The unique combination of the ligninolytic peroxidases and laccases to the white-rot fungi may be significant in that these fungi are the most efficient among the woodrotting fungi at lignin degradation.
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2
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Peroxidases The mechanism of oxidation applicable to all peroxidases, shown in Figure 1A, has been reviewed in detail for the fungal peroxidases (5). The ferric form of a peroxidase is oxidized by H 0 via two electron transfer. The oxidized enzyme can then perform two sequential, one-electron, direct oxidations of reductants that interact with the peroxidase. The fungal peroxidases are unique in that they are strong oxidants with reduction potentials of 1.4 volts (V) (
