Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved 509VSG511 tripeptide

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Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved 509VSG511 tripeptide

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Publication Article, peer reviewed scientific
Title Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: the connection between the C-terminal plug and the conserved 509VSG511 tripeptide
Author(s) Zumarraga, Miren ; Dominguez, Cristina Vaz ; Camarero, Susana ; Shleev, Sergey ; Polaina, Julio ; Martinez-Arias, Arturo ; Ferrer, Manuel ; De Lacey, Antonio ; Fernandez, Victor ; Ballesteros, Antonio ; Plou, Francisco ; Alcalde, Miguel
Date 2008
English abstract
A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial satn. 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 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The KmO2 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 509VSG511 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.
Host/Issue Combinatorial Chemistry & High Throughput Screening;10
Volume 11
ISSN 1386-2073
Pages 807-816
Language eng (iso)
Subject(s) Ascomycete laccases
C-terminal plug
combinatorial saturation mutagenesis
redox potential
Saccharomyces
Medicine
Research Subject Categories::MEDICINE::Chemistry::Biochemistry
Handle http://hdl.handle.net/2043/6858 (link to this page)

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