Incorporation of Copper Ions into T2/T3 Centers of Two-Domain Laccases

Investigations of Accessibility of T2/T3 Copper Center of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

“…The structures of SgfSL, and its mutant forms with sufficient occupancy of copper ions, were obtained via the cultivation of the corresponding over-producer, the E. coli strain, in a medium with a high concentration of copper(II) sulfate (1 mM); in the case of cultivation with 0.25 mM CuSO 4 , the laccase crystal structures were deprived of copper (Table 2) [10].…”

“…Interestingly, not only Cu2 but also Cu3 β were absent from the structure of this mutant (PDB entry 5MKM). Recently, we have shown that the T3 β of SgfSL is accessible for copper ions during dialysis of the protein preparation [10]. We propose that shuttering the T3 β tunnel in the His165Phe mutant may impede copper ion access to the TNC.…”

“…The pQE-30-based plasmid pQE-993nS carrying the gene encoding SgfSL deprived of the N-terminal signal sequence [10] was used as a template for the PCR with a pair of corresponding mutagenic primers (replaced nucleotides are in italic and underlined). Herein and after, the N-terminal truncated SgfSL will be referred to as the wild type (SgfSL wt) as the truncation does not affect the catalytic activity.…”

“…The structures were determined by molecular replacement with Phaser [33] using the structure of a mutant laccase from S. griseoflavus determined at 1.9 Å resolution (PDB entry 5O4Q [10]) as a search model. Water molecules and metal ions were removed from the model.…”

“…We determined three-dimensional structures of recombinant 2D laccases from Streptomyces viridochromogenes Ac-629 [8] and S. griseoflavus Ac-993 (SgfSL) [9]. Comparative structural analysis of TNC environments of 3D laccases and 2D laccases showed differences in substrate/product transport network, connecting T2/T3 center with surface of the protein [10]. In 3D laccases wide, clearly defined tunnels between the surface and the trinuclear site provide access to the center during the catalytic cycle.…”