Properties of Fungal Laccases

Ligninolytic white-rot fungi produce high amounts of laccases and usually excrete several isoforms of the enzyme (Blaich and Esser 1975; Bollag and Leonowicz 1984; Baldrian 2006). Depending on the species, the addition of copper (Palmieri et al. 2000; Galhaup and Haltrich 2001), sugars and amino acids (Sandhu and Arora 1985), ethanol (Lomascolo et al. 2003), and phenolic compounds such as 2,5-xylidine (Sandhu and Arora 1985; Fahreus and Reinhammar 1967) increase the production of extracellular laccases or induce the secretion of additional isoenzymes into the culture medium. Fungal laccases are glycosylated, usually in the range between 10 and 25 mol%. The glucans consist of arabinose, xylose, mannose, galactose and glucose units, which are N-linked to the polypeptide. Glycosylation may protect laccases from proteolytic degradation in the environment.

The mean optimum reaction temperature is around 55°C, although the thermostability of fungal laccases varies considerably. The half-life at 50°C ranges from minutes in Botrytis cinerea to over 3 h in Lentinus edodes and Agaricus bisporus and up to 70 h in Trametes sp. Typical fungal laccases have a molecular mass of 60-70 kDa and an acidic isoelectric point around pH 4.0. The amino acid chain contains about 520-500 amino acids, starting with an N-terminal secretion peptide (Gianfreda et al. 1999; Baldrian 2006).

Laccase is a prominent member of the blue multicopper oxidase family, which have four copper ions in their polypeptide chains (Table 13.1). The T1 copper has a trigonal coordination, with two histidines and a cysteine as conserved ligands, while one position is usually variable. It is the site of substrate oxidation and it has been widely argued that this axial ligand strongly influences the oxidation potential of the enzyme, which varies between E +400 and +800 mV, depending on the individual laccase (Xu et al. 1996; Shleev et al. 2005). The T2 and T3 copper atoms form a trinuclear cluster, where the reduction of molecular oxygen to water takes place. The T2 copper is coordinated by two histidines and one water molecule, and each of the two T3 copper atoms by three histidines. Some laccase variants lack the T1 copper and are often referred to as the "yellow laccases," as they show no characteristic absorption band around 600 nm (Leontievsky et al. 1997).

The crystal structures of the fungal laccases from Coprinus cinerius (Ducros et al. 1998), Melanocarpus albomyces (Hakulinen et al. 2002), Trametes versicolor

(Antorini et al. 2002; Bertrand et al. 2002a, b), Pycnoporus cinnabarinus (Antorini et al. 2002), and Rigidoporus lignosus (Garavaglia et al. 2004) have been resolved. They show that the protein monomer is organized into three sequentially arranged cupredoxin domains. All three domains display a similar ß-barrel type architecture that is related to those of smaller blue copper proteins such as azurin or plastocyanin. Disulfide bonds link domain one with domains two and three, while the trinuclear cluster bridges the first and third domains. The T1 copper located in domain three is the primary substrate electron acceptor site and is connected to the oxygen-reducing T2/T3 trinuclear cluster by a His-Cys-His tripeptide. Although usually active as monomeric proteins, some laccases consist of several subunits, forming hetero- (Yaver et al. 1996) or homodimers (de Souza and Peralta 2003).

Prokaryotic laccase enzymes have a similar structure (Enguita et al. 2003), although only two cupredoxin domains have been found for the bacterial laccases of Streptomyces griseus (Endo et al. 2003), which is active as a homotrimer, and S. coelicolor (Machczynski et al. 2004; Skalova et al. 2007).

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