title: 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 kcat, Km, and kcat/Km 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.
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors: Manuel
authors: M
authors: Maestre-Reyna
authors: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
authors: Wei-Chun
authors: WC
authors: Liu
authors: Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors: Wen-Yih
authors: WY
authors: Jeng
authors: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors: Cheng-Chung
authors: CC
authors: Lee
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors: Chih-An
authors: CA
authors: Hsu
authors: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
authors: Tuan-Nan
authors: TN
authors: Wen
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan; Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
authors: Andrew H-J
authors: AH
authors: Wang
authors: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan; Ph.D. Program for Translational Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors: Lie-Fen
authors: LF
authors: Shyur
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D006031:Glycosylation; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020074:Lentinula / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D042845:Laccase