The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair.
Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.
Garavaglia
Silvia
S
DiSCAFF-INFM, University of Piemonte Orientale A. Avogadro, 28100 Novara, Italy.
Cambria
Maria Teresa
MT
Miglio
Marco
M
Ragusa
Santa
S
Iacobazzi
Vito
V
Palmieri
Ferdinando
F
D'Ambrosio
Chiara
C
Scaloni
Andrea
A
Rizzi
Menico
M
D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014867:Water
D018076:DNA, Complementary; D008024:Ligands; D014867:Water; D003300:Copper; D042845:Laccase; D010100:Oxygen