Combinatorial Saturation Mutagenesis of the Myceliophthora thermophila Laccase T2 Mutant: the Connection between the C-Terminal Plug and the Conserved VSG Tripeptide

Author(s): Miren Zumarraga, Cristina Vaz Dominguez, Susana Camarero, Sergey Shleev, Julio Polaina, Arturo Martinez-Arias, Manuel Ferrer, Antonio L. De Lacey, Victor M. Fernandez, Antonio Ballesteros, Francisco J. Plou, Miguel Alcalde.

Journal Name: Combinatorial Chemistry & High Throughput Screening

Volume 11 , Issue 10 , 2008

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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 VSG tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The K 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 VSG 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.

Keywords: Ascomycete laccases, C-terminal plug, combinatorial saturation mutagenesis, redox potential, Saccharomyces cerevisiae

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Article Details

VOLUME: 11
ISSUE: 10
Year: 2008
Page: [807 - 816]
Pages: 10
DOI: 10.2174/138620708786734235
Price: $58

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