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

摘要: A mutant laccase from the Ascomycete Myceliophthora thermophila has been submitted to iterative cycles of combinatorial saturation mutagenesis through in vivo overlap extension Saccharomyces cerevisiae. Over 180,000 clones were explored, among which S510G revealed a direct interaction between conserved VSG tripeptide, located neighborhood T1 site, and C-terminal plug. The K value increased 1.5-fold, electron transfer pathway reducing substrate copper ion was altered, improving catalytic efficiency towards non-phenolic phenolic substrates by about 3- 8-fold. Although geometry at site perturbed mutation, paradoxically redox potential not significantly altered. Together, results obtained this study suggest that tripeptide may play hitherto unrecognized role regulating traffic oxygen plug, latter blocking access T2/T3 cluster native enzyme.