Abstract
The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.
Highlights
White rot fungi degrade the aromatic polymer lignin with extracellular oxidative enzymes
Mutation of a Trp residue (W171S) in the alternate proposed substratebinding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators
For lignin peroxidase (LP), the secondary metabolite, veratryl (3,4-dimethoxybenzyl) alcohol has been proposed to play a similar role [15]. This alcohol has been shown to facilitate the oxidation of many compounds in vitro (16 –18). This observation led these investigators to speculate that the veratryl alcohol cation radical is the low molecular weight mediator in the case of LP
Summary
White rot fungi degrade the aromatic polymer lignin with extracellular oxidative enzymes. These enzymes include peroxidases and/or laccases [1, 2]. LP oxidizes a variety of phenolic and nonphenolic aromatic compounds [10, 11] How these two enzymes interact with their ultimate substrate, lignin, has been intensely investigated. For LP, the secondary metabolite, veratryl (3,4-dimethoxybenzyl) alcohol has been proposed to play a similar role [15] This alcohol has been shown to facilitate the oxidation of many compounds in vitro (16 –18). Residues Ile, Val184, Gln222, Phe148, His, Glu146, and Asp183 are located in this channel This channel is sterically restricted and would not allow access to large bulky (lignin) substrates. Site-directed mutagenesis studies where Glu146 and Trp171 were altered indicate that the site of electron transfer is Trp171
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