title: Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.
abstract: The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki, P. O. Box 56, 00014 Helsinki, Finland. Kristiina.s.hilden@helsinki.fi
authors.forename: Kristiina
authors.initials: K
authors.lastname: Hildén
authors.forename: Miia R
authors.initials: MR
authors.lastname: Mäkelä
authors.forename: Taina
authors.initials: T
authors.lastname: Lundell
authors.forename: Jaana
authors.initials: J
authors.lastname: Kuuskeri
authors.forename: Alexey
authors.initials: A
authors.lastname: Chernykh
authors.forename: Ludmila
authors.initials: L
authors.lastname: Golovleva
authors.forename: David B
authors.initials: DB
authors.lastname: Archer
authors.forename: Annele
authors.initials: A
authors.lastname: Hatakka
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D016415:Sequence Alignment
chemical_list: D005656:Fungal Proteins; D042845:Laccase