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

    65 Citations (Scopus)

    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

    Keywords

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

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