Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 M. The LiP-DHP interaction was controlled by the ionization group with a pK a of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A oneelectron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-f low technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 M, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s ؊1 , respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This HisAsp⅐⅐⅐proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.Lignin is the most abundant renewable aromatic polymer and is known as one of the most recalcitrant biomaterials on earth (1, 2). Its degradation plays a key role in the carbon cycle of the biosphere (2-7). Only white-rot basidiomycetes are responsible for the complete mineralization of this polymer. Phanerochaete chrysosporium, the best studied white-rot fungus, secretes two heme peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP) under ligninolytic conditions (3-8). Thus, these enzymes have been believed to be involved in triggering lignin biodegradation. MnP oxidizes Mn II to Mn III , and the latter acts as a freely diffusible one-electron oxidizer, nonspecifically reacting with terminal organic substrates such as phenols, thiols, and lignin (3, 8 -12). This nonspecific manner is advantageous for lignin degradation because lignin is such a heterogeneous polymer.LiP is another unique heme peroxidase secreted by P. chrysosporium. It catalyzes a one-electron oxidation of nonphenolic aromatic compounds, forming the aryl cation radical (13,14), suggesting that oxidized intermediates of the enzyme possess a very high redox potential. The mechanism of LiP catalytic action on lignin is still uncertain, because it has not been clear whether LiP can oxidize lignin through a direct interaction or through radical mediation. Veratryl (3,4-dimethoxybenzyl) alcohol (VA), a preferred substrate for LiP, is synthesized de novo by P. chrysosporium under ligninolytic conditions (15). The...
A novel hydrogen peroxide-dependent phenol oxidase (TAP) was isolated from the basidiomycete Termitomyces albuminosus. TAP is an extracellular monomeric enzyme with an estimated molecular weight of 67 kDa. The purified enzyme can oxidize various phenolic compounds in the presence of hydrogen peroxide, but cannot oxidize 3,4-dimethoxybenzyl (veratryl) alcohol. Mn(II) was not required for catalysis by TAP. The optimum pH for TAP activity was 2.3, which is the lowest known optimum pH for a fungal phenol oxidase. The cDNA encoding TAP was cloned with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers based on the N-terminal amino acid sequence of TAP and 5' rapid amplification of cDNA ends (RACE)-PCR. The cDNA encodes a mature protein of 449 amino acids with a 55-amino-acid signal peptide. The deduced amino acid sequence of TAP showed 56% identity with dye-decolorizing heme peroxidase (DYP) from the ascomycete Geotrichum candidum Dec 1, but no homology with other known peroxidases from fungi.
Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.
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