Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure

Martina Andberg (Corresponding Author), Nina Hakulinen, Sanna Auer, Markku Saloheimo, Anu Koivula, Juha Rouvinen, Kristiina Kruus

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.
Original languageEnglish
Pages (from-to)6285-6300
Number of pages16
JournalFEBS Journal
Volume276
Issue number21
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Laccase
Yeast
Saccharomyces cerevisiae
Enzymes
Amino Acids
Crystal structure
Mutagenesis
Ascomycota
Proteins
Trichoderma
Site-Directed Mutagenesis
Copper
Hydrogen
Tunnels
Hydrogen bonds
Complementary DNA
Defects
Mutation
Kinetics
Geometry

Keywords

  • ascomycete
  • C-terminal plug
  • multicopper oxidase
  • mutants
  • site-directed mutagenesis

Cite this

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title = "Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure",
abstract = "The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.",
keywords = "ascomycete, C-terminal plug, multicopper oxidase, mutants, site-directed mutagenesis",
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language = "English",
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Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure. / Andberg, Martina (Corresponding Author); Hakulinen, Nina; Auer, Sanna; Saloheimo, Markku; Koivula, Anu; Rouvinen, Juha; Kruus, Kristiina.

In: FEBS Journal, Vol. 276, No. 21, 2009, p. 6285-6300.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure

AU - Andberg, Martina

AU - Hakulinen, Nina

AU - Auer, Sanna

AU - Saloheimo, Markku

AU - Koivula, Anu

AU - Rouvinen, Juha

AU - Kruus, Kristiina

PY - 2009

Y1 - 2009

N2 - The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.

AB - The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.

KW - ascomycete

KW - C-terminal plug

KW - multicopper oxidase

KW - mutants

KW - site-directed mutagenesis

U2 - 10.1111/j.1742-4658.2009.07336.x

DO - 10.1111/j.1742-4658.2009.07336.x

M3 - Article

VL - 276

SP - 6285

EP - 6300

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 21

ER -