Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved (509)VSG(511) tripeptide.
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.
Zumárraga
Miren
M
Department of Biocatalysis, Institute of Catalysis, Consejo Superior de Investigaciones Cientificas, Cantoblanco, Madrid, Spain.
Vaz Domínguez
Cristina
C
Camarero
Susana
S
Shleev
Sergey
S
Polaina
Julio
J
Martínez-Arias
Arturo
A
Ferrer
Manuel
M
De Lacey
Antonio L
AL
Fernández
Victor M
VM
Ballesteros
Antonio
A
Plou
Francisco J
FJ
Alcalde
Miguel
M
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
D010455:Peptides; D042845:Laccase