title: Role of the C-terminus of Pleurotus eryngii Ery4 laccase in determining enzyme structure, catalytic properties and stability.
abstract: The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.
authors.affiliation: CNR-Istituto di Scienze delle Produzioni Alimentari (ISPA), Lecce, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bleve
authors.forename: Chiara
authors.initials: C
authors.lastname: Lezzi
authors.forename: Stefano
authors.initials: S
authors.lastname: Spagnolo
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tasco
authors.forename: Maria
authors.initials: M
authors.lastname: Tufariello
authors.forename: Rita
authors.initials: R
authors.lastname: Casadio
authors.forename: Giovanni
authors.initials: G
authors.lastname: Mita
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rampino
authors.forename: Francesco
authors.initials: F
authors.lastname: Grieco
mesh_terms: D055162:Biocatalysis; D003001:Cloning, Molecular; D019295:Computational Biology; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D020076:Pleurotus / Q000201:enzymology*; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007527:Isoenzymes; D042845:Laccase