Purification, crystallization and preliminary X-ray study of the fungal laccase fromCerrena maxima

Abstract

PDB Reference: laccase, 2h5u, r2h5usf.Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; R free = 23.835; r.m.s.d. bond lengths, 0.06 Å ; r.m.s.d. bond angles, 1.07 ) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO 2 substituents.

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase

“…Additionally, a depleted T2 Cu was found in the pH 4.0 structure: no electron density was observed in the site corresponding to T2 Cu in F o À F c maps at 3.0. Partial occupancy of this site has been commonly observed in crystal structures of several BMCOs deposited in the PDB (entries 2h5u, 3gdc, 2zwn, 2bhf and 1uvw; Lyashenko et al, 2006;I. S. MacPherson, W. C. Lee, T. I. Liang & M. E. P. Murphy, unpublished work;Komori et al, 2009;Bento et al, 2005;Enguita et al, 2004), but complete depletion has previously only been reported in C. cinereus laccase structures (PDB entries 1a65 and 1hfu; Ducros et al, 1998Ducros et al, , 2001).…”

Kinetic analysis and structural studies of a high‐efficiency laccase from Cerrena sp. RSD1

“…The protein sequence and crystal structure of DLac were compared with the known structures of basidiomycete laccases, including TvLac and laccases from Trametes trogii , Coriolopsis gallica , Lentinus sp. , Trametes hirsute , and Cerrena maxima (Figs ). The phylogenetic tree using the primary amino acid sequence of DLac locates it far from other basidiomycete laccases (Fig.…”

Force Field Parameters for 3-Nitrotyrosine and 6-Nitrotryptophan

“…5 Despite the accumulated evidence for protein nitration, its biological consequences are not well understood on a molecular level primarily due to scarcity of the 3D structures of nitrated proteins. So far crystal structures have been solved only for a few proteins with a nitrated tyrosine [6][7][8][9] and none with a nitrated tryptophan. To overcome the limited structural information on nitrated proteins, we employed a computational approach to probe the structural perturbations induced by protein nitration.…”

“…Since torsional degrees of freedom are an important determinant of the protein conformation, we need to estimate their force constants very accurately. X-ray structures of proteins containing NIY residues show a rotation about the C-NO2 bond 6,7,9 as well as an outof-plane deformation 6 of the nitro group. Therefore we decided to obtain refined force constants for both the proper dihedral CA-CA-NO-O2 (τCCNO) and improper dihedral CA-O2-NO-O2 (τCONO).…”

“…So far crystal structures have been solved only for a few proteins with a nitrotyrosine, i.e., Cu,Zn-superoxide dismutase (CuZn-SOD), 6 MnSOD, 7 glutathione reductase, 8 and laccase. 9 Structures of nitrotryptophan-containing proteins are not available yet. We were not able to carry out MD simulations on these proteins, however, since some parameters are not provided in the AMBER-99 force field (for example, deprotonated histidine anion in CuZnSOD, Mn in MnSOD, FAD in glutathione reductase, and glycosylation in laccase).…”