Based on their structural and mechanistic properties, laccases belong to the multicopper oxidase family, along with bilirubin oxidase, ascorbate oxidase, ceruplasmin, and other homologs including those found in bacterial spore coat and copper resistance/efflux machinery [14, 15]. Numerous laccases have been cloned or are known for genome sequences. The conserved 10 histidines (His) and one cysteine (Cys) residues enable these proteins to possess four catalytic copper atoms, conventionally classified into three types according to the copper's coordination and spectroscopic properties.

The extracellular fungal laccase is archetypical. In general, a fungal laccase has a molecular mass of -60-80 kDa and an isoelectric point pi of ~4-7, depending on glycosylation [1]. Of the four copper ions, the type 1 Cu (T1, ligated by at least one Cys and two His) has a strong electronic absorption around 600 nm (its

Table 2.1 General molecular properties of typical laccases.

Source kDa pI Glycosylation Cu

Fungal -60-80 ~4-7 -5-30% Bacterial -20-80 ~6-8 None

4/subunit -20 4/subunit -10-20

relative intensity to that at 280 nm, A280/A600, may be used to gauge the purity of the resting laccase) and a characteristic EPR signal. The type 2 Cu (T2, ligated by two His) also has a characteristic EPR signal, and the strongly coupled type 3 Cu pair (T3, each ligated by three His) has a weak UV absorption around 330 nm and is EPR active only when perturbed by strong anion binding [13].

Being an oxidoreductase, laccase's Cu sites have defined redox potentials (E °). For the T1 and T3 Cu, some laccases have a "low" E ° of -0.4-0.5 V (versus the normal hydrogen electrode), while others have a "high" E° of -0.7-0.8 V [8]. However, the E ° of the T2 Cu appears to be -0.4 V for both the low- and high-E° laccase groups [16]. Table 2.1 summarizes the general molecular properties of laccases that are often relevant for their applications.

High-resolution crystallographic structures of a few fungal and bacterial lac-cases, including those of certain enzyme-substrate complexes, have recently been determined [17-22]. In general, three distinct domains in the folding of the backbone polypeptide chain are found. Combined with protein engineering work [23-27], the structural insight gained could enable us to further understand and engineer laccases.

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