## 5 Structural Properties of Mushroom Laccases  
For lignin depolymerization, most mushroom laccases are secreted dimeric or tetrameric glycoproteins that contain signal sequences, which are cleaved during a protein secretion process via the productive folding of glycoproteins in the endoplasmic reticulum (ER) [135]. The molecular weights of monomeric fungal lacases range from 30 to 130 kDa (Table 1). The apparent sizes of the proteins are usually greater than predicted theoretical molecular weights based on the calculation of their protein sequences due to protein N-glycosylation. The overall sequence homologies of fungi lacase proteins are less than 30%. Nevertheless, the copper-binding motifs and the overall structures are well-conserved and share several residues close to the metal-binding site (Figure 3a). Laccases have four copper-binding motifs conserved: NH\({}_{2}\)-His-x-His-Gly-CO\({}_{3}\) for Cu motif I, NH\({}_{2}\)-His-x-His-CO\({}_{3}\)\({}^{-}\) for Cu motif II, NH\({}_{2}\)-His-x-x-His-x-His-CO\({}_{3}\)\({}^{-}\) for Cu motif III, and NH\({}_{2}\)-His-Cys-His-x-x-His-x-x-Met/Leu/Phe-CO\({}_{3}\)\({}^{-}\) for Cu motif IV (where x represents any amino acid). A protein structural analysis of mushroom laccase revealed that the active sites consist of four copper centers with four different catalytic forms (T1, T2, T3, and T2/T3), which are arranged with the identified residues of the motifs for the interaction of copper atoms [136, 137] (Figure 3b). The T1 copper atom is bound by Cys, Met, and His residues in the motif IV and another His residue in motif III, with a distance of 2.05-3.27 A. This T1 copper-binding protein or domain is called cupredoxin. The T2 copper atom of the trinuclear center is coordinated by two His-residues in motifs I and III, with a distance of less than 2.0 A. The other two T3 copper atoms have interactions with three His-residues in motifs I--IV, with a distance of about 2.0 A. In addition, the three T2/T3 copper atoms are arranged in a triangular fashion. Six His-residues in the copper-binding motifs coordinate the T3 copper pair, while T2 is coordinated by the other two His residues in the motifs. The four residue-bound T1 copper atoms are distant from the T3 copper atom consisting of the trinuclear center with a distance of around 13 A. These copper ions binding in the active sites are classified as T1, T2, and T3 according to their different spectroscopic characteristics with the signals of electronic paramagnetic resonance (EPR) [136]. The oxidation mechanism of laccase occurs in its four copper centers via three main steps: the enzyme initially oxidizes a substrate at a mononuclear copper center T1 or blue copper center. Then, electrons are transferred internally from the T1 copper atom at a distance of around 13 A though a Cys-His pathway to the tri-nuclear copper center, a normal type (T2), and a binuclear type (T3), where dioxygen is reduced by four electrons, generating two water molecules. In the laccase reaction, the monoelectronic oxidation of  
Figure 3: Sequence alignment (**a**) and putative structural model (**b**) of fungal laccases. **(a**) Partial sequence alignment of _Trametes versicolor_ laccase and its homologs. PDB accession number: 1 KYA, _Trametes versicolor_ laccase; 2 HZH, _T. ochracea_ laccase; 1 GYC, _T. versicolor_ laccase 2; 3T6V, _Stecherinum ochracea_ laccase; 1A65, _Coprinus cinerea_ laccase; 5 EHF, _Antroidella figinea_ laccase; 1V10, _Rigidoporus microporous_ laccase; 3 PPS, _Cnaaromyces arenarius_ laccase; 2Q9O, _Melancarplus albomyces_ laccase-1. Four copper (Cu) binding motifs were marked. (**b**) Overall structure of _Hericium erinaceous_ laccase 1 and a closer view of the active site for putative copper binding in a laccase with modification from the reference [137]. The mushroom increase structure was predicted by PHYRE2 based on the homologous lactose 3-D structure in Pdb data files. substrates generates free radicals.