PMID:22526780

Citation	Hildén, K, Mäkelä, MR, Lundell, T, Kuuskeri, J, Chernykh, A, Golovleva, L, Archer, DB and Hatakka, A (2013) Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus. Appl. Microbiol. Biotechnol. 97:1589-99
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.
Links	PubMed Online version:10.1007/s00253-012-4011-6
Keywords	Amino Acid Sequence; Basidiomycota/chemistry; Basidiomycota/enzymology; Basidiomycota/genetics; Cloning, Molecular; Enzyme Stability; Fungal Proteins/chemistry; Fungal Proteins/genetics; Fungal Proteins/metabolism; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Laccase/chemistry; Laccase/genetics; Laccase/metabolism; Models, Molecular; Molecular Sequence Data; Pichia/genetics; Pichia/metabolism; Protein Engineering; Sequence Alignment
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9APHY:I1W1V8		GO:0052716: hydroquinone:oxygen oxidoreductase activity	ECO:0000315:		F		Fig. 5	complete CACAO 8859

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