Structural and functional roles of glycosylation in fungal laccase from lentinus sp.

Manuel Maestre-Reyna, Wei Chun Liu, Wen Yih Jeng, Cheng Chung Lee, Chih An Hsu, Tuan Nan Wen, Andrew H.J. Wang, Lie Fen Shyur

Research output: Contribution to journalArticle

29 Citations (Scopus)

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.

Original languageEnglish
Article numbere0120601
JournalPloS one
Volume10
Issue number4
DOIs
Publication statusPublished - 2015 Apr 7

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Lentinula
Lentinus
Glycosylation
Laccase
laccase
glycosylation
Pichia pastoris
Enzymes
kinetics
Kinetics
enzymes
copper
lignosulfonates
inorganic compounds
Inorganic compounds
Basidiomycota
mutants
white-rot fungi
Peptides
Calorimetry

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Maestre-Reyna, M., Liu, W. C., Jeng, W. Y., Lee, C. C., Hsu, C. A., Wen, T. N., ... Shyur, L. F. (2015). Structural and functional roles of glycosylation in fungal laccase from lentinus sp. PloS one, 10(4), [e0120601]. https://doi.org/10.1371/journal.pone.0120601
Maestre-Reyna, Manuel ; Liu, Wei Chun ; Jeng, Wen Yih ; Lee, Cheng Chung ; Hsu, Chih An ; Wen, Tuan Nan ; Wang, Andrew H.J. ; Shyur, Lie Fen. / Structural and functional roles of glycosylation in fungal laccase from lentinus sp. In: PloS one. 2015 ; Vol. 10, No. 4.
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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 {\AA} 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.",
author = "Manuel Maestre-Reyna and Liu, {Wei Chun} and Jeng, {Wen Yih} and Lee, {Cheng Chung} and Hsu, {Chih An} and Wen, {Tuan Nan} and Wang, {Andrew H.J.} and Shyur, {Lie Fen}",
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Maestre-Reyna, M, Liu, WC, Jeng, WY, Lee, CC, Hsu, CA, Wen, TN, Wang, AHJ & Shyur, LF 2015, 'Structural and functional roles of glycosylation in fungal laccase from lentinus sp.', PloS one, vol. 10, no. 4, e0120601. https://doi.org/10.1371/journal.pone.0120601

Structural and functional roles of glycosylation in fungal laccase from lentinus sp. / Maestre-Reyna, Manuel; Liu, Wei Chun; Jeng, Wen Yih; Lee, Cheng Chung; Hsu, Chih An; Wen, Tuan Nan; Wang, Andrew H.J.; Shyur, Lie Fen.

In: PloS one, Vol. 10, No. 4, e0120601, 07.04.2015.

Research output: Contribution to journalArticle

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T1 - Structural and functional roles of glycosylation in fungal laccase from lentinus sp.

AU - Maestre-Reyna, Manuel

AU - Liu, Wei Chun

AU - Jeng, Wen Yih

AU - Lee, Cheng Chung

AU - Hsu, Chih An

AU - Wen, Tuan Nan

AU - Wang, Andrew H.J.

AU - Shyur, Lie Fen

PY - 2015/4/7

Y1 - 2015/4/7

N2 - 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.

AB - 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.

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