Crystal Structure of a Laccase from the FungusTrametes versicolor at 1.90-Å Resolution Containing a Full Complement of Coppers

Abstract

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O 2 reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

“…The starting geometry of the trinuclear Cu site was adapted from the crystal structure of Trametes versicolor Lc (1GYC, Res. 1.9 Å) (29), in which the His ligands were replaced by imidazolyl ligands. To fully reflect the features of the crystal structure, (i) the positions of the H atoms that replaced the side chains to the protein backbone and those bound to His N not bound to Cu (all involved in hydrogen bonds) were fixed; (ii) the angle of the O atom on the T2 water-derived ligand relative to the plane of the two T2 His rings was fixed to prevent it from artificial binding to the nearby T3 His ligands; and (iii) the three N-Cu T3␣ -Cu T3␤ -N dihedral angles, where N is the coordinated atom of the eclipsed His ligand, were fixed to keep the eclipsed conformation as found in the crystal structures of all MCOs.…”

“…A Glu residue near the T3 site appears to play a significant role in stabilizing the structure found in the AO and CotA crystals, because it is well positioned for hydrogen bonding with the azide(s), directly or via water molecules. This Glu (E 510 in AO and E 498 in CotA) is in the putative solvent channel (19,29), and the azide may well have been trapped by this residue in the crystals (prepared by soaking the resting crystals in azide solution). Notably, DFT calculations indicate that the NI Az structure is lower in energy than the T3 -1,1 end-on bridged structure of CotA (SI Fig.…”

Cloning and characterization of the genes encoding the high-affinity iron-uptake protein complex Fet3/Ftr1 in the basidiomycete Phanerochaete chrysosporium

“…pombe (37.1 %). The alignment and the manual examination of the Pc-Fet3 coding sequence revealed that the catalytic histidines participating in the copper centres (Piontek et al, 2002) are encoded in exons II, III, V and VI. In contrast, in Pc-MCO1, these histidines are encoded in exons III, V, VI, VII, XVI, XIX and XX.…”

“…The crystal structure of the Sac. cerevisiae Fet3p and its subsequent superposition with laccase from Trametes versicolor (Piontek et al, 2002) showed several differences in the surroundings of the aromatic-substrate-binding pocket observed in laccases (Taylor et al, 2005). It has been generally stated that Fet3p and ceruloplasmin are the only members of the MCO family that show ferroxidase activity.…”

“…Ceruloplasmin is the most complex member of this group, with a six-domain structure possessing three T1 copper centres plus one T2/ T3 cluster (Zaitsev et al, 1999). Ascorbate oxidase is a dimer composed of two laccase-like monomers (Messerschmidt et al, 1992), while laccase is the simplest member of the family, containing only one T1 and one T2/ T3 centre (Piontek et al, 2002). In this regard, ferroxidases are similar to laccases, since they also have only one T1 and one T2/T3 copper centre.…”