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An extracellular lignin-degrading enzyme from the basidiomycete Phanerochaete chrysosporium Burdsall was purified to homogeneity by ion-exchange chromatography. The 42,000-dalton ligninase contains one protoheme IX per molecule. It catalyzes, nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds:Ca-Cp cleavage in lignin model compounds of the type aryl-C.HOH-CpHR-CyH20H (R = -aryl or -0-aryl), oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in phenylglycol structures, and hydroxylation of benzylic methylene groups. It also catalyzes oxidative coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. All reactions require H202. The Ci-Cp cleavage and methylene hydroxylation reactions involve substrate oxygenation; the oxygen atom is from 02 and not H202. Thus the enzyme is an oxygenase, unique in its requirement for H202.We recently reported the discovery of a lignin-degrading enzyme from the basidiomycete Phanerochaete chrysosporium Burdsall (Aphylophorales, Corticiaceae) (1). This ligninase is extracellular and requires H202 for activity. This paper describes its purification and characterization.The enzyme catalyzes C-C bond cleavage in the propyl side chains of two dimeric model compounds, as well as in spruce and birch lignins (1). This cleavage is prominent in the fungal degradation of lignin (2) and is the first reaction in the metabolism of dimeric models in cultures (3, 4). The studies here reveal that the enzyme is a heme-containing oxygenase, unique in that it requires H202. To study specific reactions we have used lignin substructure model compounds as substrates, rather than lignin. The two types of models chosen are of the 13-1 (1,2-diarylpropane-1,3-diol) and P-0-4 (arylglycerol-,B-aryl ether) types. Together, the /3-1 and P-0-4 linkages, represented by models I and II, respectively ( Fig. 1), make up over 60% of the intermonomer linkages in lignins (5). MATERIALS AND METHODSEnzyme Production and Purification. P. chrysosporium, strain BKM-1767 (ATCC 24725), was maintained and spore inoculum was prepared and used as reported previously (6).The 10-ml cultures in 125-ml Erlenmeyer flasks were grown as described (7), with 10 mM 2,2-dimethylsuccinate, pH 4.5, as buffer. Enzyme activity appeared 3-4 days after culture initiation, was maximal in 6-day-old cultures, and was associated only with the extracellular culture fluid.Cultures (130, 6-day) were combined and centrifuged (10,000 x g, 15 min, 40C). To minimize proteolysis, p-methylsulfonyl fluoride (0.2 mM) (Sigma) was added to the supernatant, which was concentrated (Amicon YM-10 filter; 10,000-dalton pore size) to 250 ml. This solution oxidized 0.16 gmol of 3,4-dimethoxybenzyl (veratryl) alcohol per min per ml (see assay procedure below). After overnight dialysis against 5 mM sodium tartrate buffer, pH 4.5, the sample was applied to a DEAE-Bio-Gel A column (1 x 16 cm) (Bio-Rad), previously equilibrated with the sa...

The extracellular fluid of ligninolytic cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium Burds. contains an enzyme that degrades lignin substructure model compounds as well as spruce and birch lignins. It has a molecular size of 42,000 daltons and requires hydrogen peroxide for activity.

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning ␤-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.cytochrome-c ͉ iron respiration ͉ protein film voltammetry ͉ electron paramagnetic resonance ͉ Shewanella

The white rot fungus Phanerochaete chrysosporium degraded DDT [1,1,-bis(4-chlorophenyl)-2,2,2-trichloroethane], 3,4,3',4'-tetrachlorobiphenyl, 2,4,5,2',-4',5'-hexachlorobiphenyl, 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocylohexane), and benzo[a]pyrene to carbon dioxide. Model studies, based on the use of DDT, suggest that the ability of Phanerochaete chrysosporium to metabolize these compounds is dependent on the extracellular lignin-degrading enzyme system of this fungus.

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The aromatic polymer lignin protects plants from most forms of microbial attack. Despite the fact that a significant fraction of all lignocellulose degraded passes through arthropod guts, the fate of lignin in these systems is not known. Using tetramethylammonium hydroxide thermochemolysis, we show lignin degradation by two insect species, the Asian longhorned beetle (Anoplophora glabripennis) and the Pacific dampwood termite (Zootermopsis angusticollis). In both the beetle and termite, significant levels of propyl side-chain oxidation (depolymerization) and demethylation of ring methoxyl groups is detected; for the termite, ring hydroxylation is also observed. In addition, culture-independent fungal gut community analysis of A. glabripennis identified a single species of fungus in the Fusarium solani/Nectria haematococca species complex. This is a soft-rot fungus that may be contributing to wood degradation. These results transform our understanding of lignin degradation by wood-feeding insects.Asian longhorned beetle ͉ Pacific dampwood termite ͉ TMAH thermochemolysis ͉ Anoplophora glabripennis ͉ Zootermopsis angusticollis L ignin plays a central role in carbon cycling on Earth. Its heterogeneous structure imparts plants with structural rigidity and also serves to protect cellulose and hemicellulose from degradation (1). Most of what is known about lignin biodegradation is from pure culture studies with filamentous basidiomycete fungi, known as white-rot and brown-rot decay. Although both white-rot and brown-rot fungal degradation have been characterized, much more is known about the white-rot system (2, 3). White-rot fungi simultaneously degrade the three major components of the plant cell wall: lignin, cellulose, and hemicellulose. Analysis of white-rot-degraded wood shows that the reactions in lignin: (i) are oxidative, (ii) involve demethylation (or demethoxylation), (iii) include side-chain oxidation at C ␣ , and (iv) involve propyl side-chain cleavage between C ␣ and C ␤ (Fig. 1) (4). In contrast to white-rot fungi, brown-rot fungi are able to circumvent the lignin barrier, removing the hemicellulose and cellulose with only minor modification to the lignin. Consequently, lignin remains a major component of the degraded plant cell wall (5). The remaining lignin is demethylated on aryl methoxy groups and contains a greater number of ring hydroxyl groups (6).Little is known about lignin degradation in complex ecosystems, such as insect guts, where a consortium of microbes may be involved in degradation rather than just a single species. Although cellulose degradation in insect guts is well documented (7,8), the fate of lignin has not clearly been demonstrated (9, 10), and it is widely accepted that insect gut systems do not have the capacity to degrade lignin (10). Although the majority of previous reports suggest that many wood-feeding insects overcome the lignin barrier by feeding on predegraded wood (11) or through exosymbiotic relationships with wood-degrading fungi (12, 13), there are species of insects...

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