Thus, T. cervina LiP appears to react with H2O2 in the same manner as in P. chrysosporium LiP and other plant and fungal peroxidases. The sequence analysis showed that T. cervina LiP lacks the click here tryptophan residue corresponding to Trp171 of P. chrysosporium LiP, which is the substrate-oxidation site on the protein surface (Doyle et al., 1998; Gelpke et al., 2002; Johjima

et al., 2002). Tryptophan residues corresponding to Trp171 have been found in all LiP homologs including VP (Martínez, 2002; Ruiz-Dueñas et al., 2009a). In T. cervina LiP, the position of Trp171 was substituted with a histidine residue, His170 (Fig. 1). However, the redox activity of the imidazole group is much lower than that of the tryptophan indole group. Pérez-Boada et al. (2005) demonstrated that the VP mutant W164H completely lost its LiP-type activity, suggesting that His170 is not a substrate-oxidation site in T. cervina LiP. A unique tyrosine residue (Tyr181) was found in T. cervina LiP. This is the first report of a LiP containing a tyrosine residue; tyrosine has not been found previously in any other LiP or VP sequences. The tyrosine residue is redox active and could be advantageous for a LiP-type oxidation involving

radical generation. In fact, it has been reported that tyrosine can act as a redox-active residue, like tryptophan, in different enzymes (Stubbe & van der Donk, 1998), and a tyrosyl radical has been detected in a VP variant W164Y (Ruiz-Dueñas et al., 2009b). Thus, Tyr181 might be the substrate-oxidation site of T. cervina LiP. To evaluate

a possible role of Tyr181, a structural model of T. cervina LiP was constructed using the moe Tacrolimus algorithm. The Cα topology and the 10 helices of T. cervina LiP were almost identical to those of P. chrysosporium LiP (Supporting Information, Fig. S1a). The partial structures of the heme cavity and calcium-binding sites in the proximal and distal regions Sinomenine were superimposable on the corresponding structures of P. chrysosporium LiP (Fig. S1b and c), indicating that the homology model was constructed with high accuracy. The T. cervina LiP model indicated that Tyr181 neighbors the 6-propionate group of heme and the phenolic side chain of Tyr181 is oriented toward the exterior (Fig. 3). These conformational details support the idea that there is an electron transfer pathway from Tyr181 to heme, enabling oxidation of bulky substrates such as lignin and cytochrome c. Also, the T. cervina LiP model showed that Tyr181 is surrounded by acidic amino acids just as Trp171 in P. chrysosporium LiP is surrounded by acidic amino acids (Fig. 3b). The acidic environment may stabilize the cation radical of veratryl alcohol as an enzyme-bound redox mediator (Choinowski et al., 1999; Ruiz-Dueñas et al., 2008) and improve the access of basic substrates, such as cytochrome c, to the oxidation site (Wariishi et al., 1994). Thus, it is likely that Tyr181 is a substrate-oxidation site in T.