Combinatorial saturation mutagenesis of the Myceliophthora thermophila laccase T2 mutant: The connection between the C-terminal plug and the conserved509VSG511 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 509VSG511 tripeptide, located in the neighborhood of the T1 site, and the C-terminal plug. The Km 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 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.This research was funded by the Spanish Ministry of Education and Science projects VEM2004-08559, CTQ2005-08925-C02-02/PPQ and CTQ2006-12097/BQU; the EU project NMP2-CT-2006-026456; a CSIC project 200580M121, the Ramón y Cajal Program, and the Swedish Research Council (Vetenskapsårdet, project ärandenummer: 621-2005-3581). MZ was supported by a fellowship from the “Gobierno Vasco” and CV by a fellowship from the FPU program of the “Ministerio de Educación y Ciencia”.Peer Reviewe