tailieunhanh - Báo cáo khoa học: Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure

The C-terminus of the fungal laccase fromMelanocarpus 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. | ỊFEBS Journal Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production catalytic properties and structure Martina Andberg1 Nina Hakulinen2 Sanna Auer1 Markku Saloheimo1 Anu Koivula1 Juha Rouvinen2 and Kristiina Kruus1 1 VTT TechnicalResearch Center of Finland Finland 2 Department of Chemistry University of Joensuu Finland Keywords ascomycete C-terminalplug multicopper oxidase mutants site-directed mutagenesis Correspondence M. Andberg VTT TechnicalResearch Center of Finland . Box 1000 FIN-02044 VTT Finland Fax 358 20 722 7071 Tel 358 20 722 5124 E-mail Website http research bic lang en Database The atomic coordinates and structure factors have been submitted to the Protein Data Bank under the accession number 3DKH Received 2 July 2009 revised 17 August 2009 accepted 28 August 2009 doi 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 Tricho-derma 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 delDSGL559 was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the

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