title: Heterologous expression of peptidyl-Lys metallopeptidase of Armillaria mellea and mutagenic analysis of the recombinant peptidase.
abstract: A method to express, purify and modify the Peptidyl-Lys metallopeptidase (LysN) ofArmillaria melleainPichia pastoriswas developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S1', it was proposed to extend the S1' binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P1'. Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S2'-S4' compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.
authors.affiliation: Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark and Center for Evolutionary Chemical Biology, Nano-Science Center, University of Copenhagen, Universitetsparken 5, 2100 København Ø, Denmark.
authors.forename: Anders S R
authors.initials: AS
authors.lastname: Ødum
authors.affiliation: Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark and.
authors.forename: Søren
authors.initials: S
authors.lastname: Østergaard
authors.affiliation: Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark and.
authors.forename: Inga
authors.initials: I
authors.lastname: Nørby
authors.affiliation: Center for Evolutionary Chemical Biology, Nano-Science Center, University of Copenhagen, Universitetsparken 5, 2100 København Ø, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Meldal
authors.affiliation: Global Research, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark and kjdo@novonordisk.com.
authors.forename: Kjeld
authors.initials: K
authors.lastname: Olesen
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D055392:Armillaria / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D057075:Enzyme Assays; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D001120:Arginine; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase
keywords: enzyme kinetics; enzyme mechanism; metalloprotease; molecular modelling; site-directed mutagenesis