Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase

The present study maps the active site of lignin peroxidase in respect to s ubstrate size using either fungal or recombinant wild type, as well as muta ted, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta -O-4 linkages. The fungal and recombinan t wild type lignin peroxidase both oxidized the tetrameric model forming fo ur products, The four products were identified by mass spectral analyses an d compared with synthetic standards. They were identified as tetrameric, tr imeric, dimeric, and monomeric carbonyl compounds. Ah four of these product s were also formed from single turnover experiments. This indicates that li gnin 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. M utation of the recombinant lignin peroxidase (isozyme H8) in the heme acces s channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidat ion of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidatio n of the tetrameric model. These results are consistent with lignin peroxid ase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.