Table 1.
Nucleotide sequences of PCR primers.
Table 2.
Data collection and refinement statistics of nLcc4.
Fig 1.
(A) The cDNA and deduced amino acid sequences of nlcc4.
(B) The sequence alignments of Lentinus sp. nlcc4 with other Basidiomycete laccases. The signal peptide and the predicted four Cu binding regions are indicated by solid and dotted lines, respectively. The confirmed N-glycosylation sites (Asn-X-Ser/Thr) based on the nLcc4 sequence are framed or labeled in red. The predicted N-glycosylation site on nLcc4 is labeled in green. Residues coordinated with coppers are highlighted in grey or labeled in blue. Residues examined by point mutation (N→D) for production of recombinant mutants are highlighted in yellow. nLcc4 (PDB #3X1B), Trametes hirsute (PDB #3FPX), Cerrena maxima (PDB #3DIV), Trametes versicolor (PDB #1KYA), Trametes ochracea (PDB #2HZH), Coriolopsis gallica (PDB #4A2E), Trametes trogii (PDB #2HRG), and Lentinus tigrinus (PDB #2QT6).
Fig 2.
Analysis of molecular weight and enzymatic activity of native and Endo H-deglycosylated Lcc4 by 10% SDS-PAGE and zymography.
(A) Heated laccases were separated and visualized by coomassie brilliant blue (CBR) staining and their relative mobility distances (Rf values) and molecular weights were calculated and calibrated with a set of molecular weight standards. (B) Unheated laccase samples in lysis buffer, in which the enzymes were not denatured and remained active, were separated and visualized by CBR staining and zymographic analysis using ABTS substrate. Protein marker (M), native Lcc4 (nLcc4, lane 1), nLcc4 treated with Endo H for 1 h (lane 2), overnight (lane 3), and with an extra supplement of 500 U Endo H in the overnight treatment (lane 4). (C) Optimal pH, optimal temperature and temperature sensitivity determination of nLcc4 and Endo H-deglycosylated Lcc4 (dLcc4). In the temperature sensitivity assay, laccase enzyme was pre-incubated at the indicated temperature for 10 min, and the residual enzymatic activity was then immediately measured. Black rhombi: nLcc4; white rhombi: dLcc4.
Table 3.
Kinetic properties of native (nLcc4) and Endo H-deglycosylated lcc4 (dLcc4)a.
Fig 3.
Expression profile of recombinant wild-type and mutant forms of Lcc4 in the cultural media of P. pastoris X33 cells.
(A) Time course of yeast host cell growth and protein expression and concentration. (B) CBR stained gel and zymogram of cultural media. For zymogram analysis, the snapshots of SDS gel were immersed in 2 mM ABTS solution for 20 and 60 s, respectively. Protein marker (M); vector (lane 1); wild-type (lane 2); N75D (lane 3); N162D (lane 4); N238D (lane 5); N458D (lane 6). (C) Relative laccase specific activity in the cultural medium of yeast cells transformed with vector only, wt, N75D, N162D, N238D, and N48D gene construct.
Fig 4.
Lignosulfonic acid hydrolysis with Lentinus sp. nLcc4 determined by ITC.
(A) Thermal power plots as a function of time by single-injection assay, (B) multiple-injection assay, and (C) Michaelis-Menten and Lineweaver-Burk plots of lignosulfonic acid reaction by nLcc4.
Fig 5.
Structure-assisted sequence alignment and stereo views of the structure of nLcc4.
(A) Protein sequence alignment of Lentinus sp. Lcc4 (PDB #3X1B) and Lentinus tigrinus (PDB #2QT6). The signal peptide of Lentinus sp. Lcc4 is highlighted in yellow. Residues in α-helices, β -strands, and loops are shown as red cylinders, blue arrows, and lines, respectively. The 310-helices are labeled as η1 to η8. The histidine residues involved in the copper coordination are highlighted in green. The glycosylated Asn residues are marked as magenta numbers. The four substrate binding pocket loops (SBPLs I-IV) and the domain connection loops (D1-D2 and D2-D3 loops) are also indicated. (B) Ribbon diagram of laccase crystal structure, domain 1 (D1), domain 2 (D2) and domain 3 (D3) of laccase are colored pink, green and blue, respectively. Two disulfide bonds (Cys106-Cys510 and Cys138-Cys226) and three N-glycosylated sites (Asn75, Asn238, and Asn458) are labeled. The licorice representation shows: copper ions in spheres with dark grey and oligosaccharides in sticks with purple carbons. The predicted substrate binding pocket loops (SBPLs I-IV) are also indicated.
Fig 6.
Stereo views of three types of cupper binding sites and glycan moieties of nLcc4.
(A) The copper atoms, one type-1 (T1), one type-2 (T2), and two type-3 (T3a and T3b), are coordinated to the surrounding histidine, cysteine and the water molecules. Protein residues are shown as a stick model, the oxygen atoms are shown in red, nitrogen in blue, sulfur in yellow, and carbon in green. The four copper ions and water (Wat1 and Wat2) molecules are represented by gray and red spheres, respectively. (B) The N-linked glycans (magenta) on Asn75, Asn238 and Asn458 and the contact residues (green) are shown as stick models. The black dashed lines indicate hydrogen bonds or interactions between N-linked glycans and surrounding amino acid residues, in which the amino acid residues 436 to 439 form a β-strand (β 15) structure and are connected to the D2-D3 loop.
Fig 7.
Molecular dynamics simulations of Lcc4 in different glycosylation states at pH 3.0 and 303 K.
All structures represent average structures along an 18 ns production MD simulation. Molecules are colored by B-factor, as calculated for the entire simulation. Blue regions indicate low B-factors, and therefore, low molecular motion, while red regions are flexible, and have high B-factors. Deep blue indicates B-factors of 10 Å2 or lower, while bright red indicates values equal to, or higher than, 50 Å2. The thickness of the represented structure is also proportional to the B-factor value. (A) Simulation of nLcc4 on the left (Video A in S2 File). On the right, the general structure of nLcc4, highlighting the original crystal structure. (B) Partially de-glycosylated laccase without the Asn75 N-glycan (Video B in S2 File). (C) Partially de-glycosylated laccase without the Asn238 N-glycan (Video C in S2 File). (D) Partially de-glycosylated laccase without the Asn458 N-glycan (Video D in S2 File). (E) dLcc4, containing only the first GlcNAc at each of the confirmed N-glycosylated sites (Video E in S2 File).
Fig 8.
Hydrogen bond network of the three glycans under different glycosylation conditions.
Hydrogen bonds are depicted as lines stretching from one partner to the other. Red lines represent strong bonds (occupancy >10% of the total simulation time) and green lines represent weak bonds (occupancy <10% of the total simulation time). Further, de-glycosylation simulation results are compared to the native state by the representation of weaker hydrogen bonds as dotted lines, and stronger hydrogen bonds with thicker lines. All data evaluation was performed with ptraj.
Fig 9.
Molecular dynamics simulations of nLcc4-lignosulfonic acid complex.
(A) Detail of the free form of nLcc4 binding pocket, corresponding to the average structure in Fig 7A. (B) Detail of the average simulated nLcc4 binding pocket bound to lignosulfonic acid (LSA), highlighting the stabilized Phe183 and Asp227 and the relative conformational changes of SBPLs I and III. (C) Full average structure of the computed nLcc4-lignosulfonic acid complex simulation. Structures represent average structures along an 18 ns production MD simulation. Molecules are colored by B-factor, as calculated for the entire simulation with the same palette as in Fig 7. The thickness of the represented structure is also proportional to the B-factor value.
Fig 10.
Comparisons of D2-D3 loop orientations.
Four bacterial laccases (PDB #2FQG, #1GSK, #2YAE and #4F7K) are superimposed with nLcc4. The D2-D3 loop in nLcc4 is threaded in direct proximity to the binding pocket; the D2-D3 loops in the other four bacterial laccases show the same orientation to connect D2 and D3 domains and pass near the N-terminus, where they cannot interact with any of the SBPLs. The D2-D3 loops from 2FQG, 1GSK, 2YAE, 4F7K and nLcc4 are colored orange, green, yellow, red and magenta, respectively.