Structural and functional roles of glycosylation in fungal laccase from lentinus sp.
Journal
PLoS ONE
Journal Volume
10
Journal Issue
4
Date Issued
2015
Author(s)
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-1 μM-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 wildtype 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. © 2015 Maestre-Reyna et al.
Other Subjects
aspartic acid; laccase; serine; threonine; laccase; recombinant protein; Article; binding site; crystal structure; deglycosylation; enzyme activity; enzyme kinetics; enzyme release; fungal gene; glycosylation; hydrogen bond; isothermal titration calorimetry; Lcc4 gene; Lentinus; molecular dynamics; nonhuman; nucleotide sequence; open reading frame; protein expression; thermostability; amino acid sequence; catalysis; chemistry; enzymology; genetics; glycosylation; kinetics; Lentinula; matrix-assisted laser desorption-ionization mass spectrometry; metabolism; molecular cloning; molecular genetics; molecular model; mutation; oxidation reduction reaction; Pichia; protein conformation; sequence homology; Fungi; Lentinus; Pichia pastoris; Amino Acid Sequence; Base Sequence; Catalysis; Cloning, Molecular; Glycosylation; Kinetics; Laccase; Lentinula; Models, Molecular; Molecular Sequence Data; Mutation; Oxidation-Reduction; Pichia; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Type
journal article