Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds

J. P. Kallio, Sanna Auer, J. Jänis, Martina Andberg, Kristiina Kruus, J. Rouvinen, Anu Koivula, N. Hakulinen (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.
Original languageEnglish
Pages (from-to)895-909
Number of pages15
JournalJournal of Molecular Biology
Volume392
Issue number4
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Laccase
Binding Sites
Mass Spectrometry
Electrons
Dimerization
Carbon Monoxide
Carboxylic Acids
Histidine
Mutagenesis
Protons
Copper
Hydrogen
Ligands

Keywords

  • 2,6-dimethoxyphenol
  • C-O dimer
  • laccase
  • mutagenesis
  • substrate binding

Cite this

Kallio, J. P. ; Auer, Sanna ; Jänis, J. ; Andberg, Martina ; Kruus, Kristiina ; Rouvinen, J. ; Koivula, Anu ; Hakulinen, N. / Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds. In: Journal of Molecular Biology. 2009 ; Vol. 392, No. 4. pp. 895-909.
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title = "Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds",
abstract = "Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.",
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Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds. / Kallio, J. P.; Auer, Sanna; Jänis, J.; Andberg, Martina; Kruus, Kristiina; Rouvinen, J.; Koivula, Anu; Hakulinen, N. (Corresponding Author).

In: Journal of Molecular Biology, Vol. 392, No. 4, 2009, p. 895-909.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds

AU - Kallio, J. P.

AU - Auer, Sanna

AU - Jänis, J.

AU - Andberg, Martina

AU - Kruus, Kristiina

AU - Rouvinen, J.

AU - Koivula, Anu

AU - Hakulinen, N.

PY - 2009

Y1 - 2009

N2 - Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

AB - Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

KW - 2,6-dimethoxyphenol

KW - C-O dimer

KW - laccase

KW - mutagenesis

KW - substrate binding

U2 - 10.1016/j.jmb.2009.06.053

DO - 10.1016/j.jmb.2009.06.053

M3 - Article

VL - 392

SP - 895

EP - 909

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 4

ER -