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.

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1,3-1,4-β-D-Glucanase (lichenase) and 1,3-β-D-glucanase (laminarinase) are fibrolytic enzymes which play an important role in the hydrolysis of polysaccharide components. Both of these glucanases have been employed in a number of industrial applications. This study aims to improve or combine the novel properties of both glucanases in an attempt to create desirable hybrid enzymes with economic benefits for industrial applications. A truncated and mutated 1,3-1,4-β-D-glucanase gene (TFs(W203F)) from Fibrobacter succinogenes, and a 1,3-β-D-glucanase gene (TmLam) from hyperthermophilic Thermotoga maritima were used as target enzymes. The substrate-binding domains (TmB₁ and TmB₂) and the catalytic domain (TmLam(CD)) of TmLam were ligated to the N- or C-terminus of TFsW203F to create four hybrid enzymes, TmB₁-TFs(W203F), TFs(W203F)-TmB₂, TmB₁-TFs(W203F)-TmB₂ and TFs(W203F)-TmLam(CD). The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB₁-TFs(W203F)-TmB₂ and TFs(W203F)-TmLam(CD), respectively. Furthermore, fluorescence and circular dichroism spectrometric analyses indicated that the hybrid TFs(W203F)-TmLam(CD) was structurally more stable than the parental TFs(W203F), which was attributed to an improved thermal tolerance of the hybrid enzyme. This study has been successful in creating bifunctional hybrid glucanases with dual substrate catalytic functions which warrant further evaluation of their possible use in industrial applications.

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