Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus sp.

Abstract

Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O2 to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic analysis revealed that the k cat, K m, and k cat/K m of nLcc4 with substrate ABTS were 3,382 s -1, 65.0 ± 6.5 μM, and 52 s -1μM-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s -1, 56.7 ± 3.2 μM, and 0.004 s -1μM-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.

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

“…This result is consistent with the study of deglycosylation and site-directed mutagenesis in Lentinus sp. laccase [12]. Both Endo H and PNGase F deglycosylation led to similar decreases in the k cat of DLac (Fig.…”

COPPER IMPROVES THE PRODUCTION OF LACCASE BY Pleurotus sajor-caju WITH ABILITY TO GROW ON EFFLUENTS OF THE CITRUS INDUSTRY

“…These changes may be due to modifications of the enzymatic structure due to changes in glycosylation patterns (Xiao et al 2006). It is known that the pattern of glycosylation affects the stability of laccase in basidiomycetes (Maestre-Reyna et al 2015). In studies conducted on eukaryotic cells, metals such as copper have been added to cell culture media and were all shown to alter the glycosylation levels of diverse proteins in unique cell lines (Yuk et al 2015).…”

A Thermo-Active Laccase Isoenzyme From Trametes trogii and Its Potential for Dye Decolorization at High Temperature

“…In addition, the optimum temperature, thermostability, and decolorizing efficiency of Lac 37 II were higher than those of the recombined LCC3 ( Table 2). The possible reasons for those differences are the posttranslational modifications in different hosts (yeasts and T. trogii), especially the glycosylation (Maestre-Reyna et al, 2015). Previous studies have proved that the recombined laccases in P. pastoris were always hyperglycosylated along with the changes of molecular mass and enzymatic properties, and the mechnism is that the glycosylation profile acts as the regulatory modules for substrate binding and turnover (Younes et al, 2007;Odón et al, 2009;Neha et al, 2012;Maestre-Reyna et al, 2015;Peter, 2016).…”