title: Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved (509)VSG(511) tripeptide.
abstract: A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension in Saccharomyces cerevisiae. Over 180,000 clones were explored, among which the S510G mutant revealed a direct interaction between the conserved (509)VSG(511) tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K(m)(O)(2) value of the mutant increased 1.5-fold, and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, improving the catalytic efficiency towards non-phenolic and phenolic substrates by about 3- and 8-fold. Although the geometry at the T1 site was perturbed by the mutation, paradoxically the laccase redox potential was not significantly altered. Together, the results obtained in this study suggest that the (509)VSG(511) tripeptide may play a hitherto unrecognized role in regulating the traffic of oxygen through the C-terminal plug, the latter blocking access to the T2/T3 copper cluster in the native enzyme.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, Consejo Superior de Investigaciones Cientificas, Cantoblanco, Madrid, Spain.
authors.forename: Miren
authors.initials: M
authors.lastname: Zumárraga
authors.forename: Cristina
authors.initials: C
authors.lastname: Vaz Domínguez
authors.forename: Susana
authors.initials: S
authors.lastname: Camarero
authors.forename: Sergey
authors.initials: S
authors.lastname: Shleev
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
authors.forename: Arturo
authors.initials: A
authors.lastname: Martínez-Arias
authors.forename: Manuel
authors.initials: M
authors.lastname: Ferrer
authors.forename: Antonio L
authors.initials: AL
authors.lastname: De Lacey
authors.forename: Victor M
authors.initials: VM
authors.lastname: Fernández
authors.forename: Antonio
authors.initials: A
authors.lastname: Ballesteros
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques; D017124:Conserved Sequence; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D010455:Peptides; D042845:Laccase