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

Zumarraga, MirenDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)

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

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.

Dominguez, Cristina VazDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Camarero, SusanaDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Shleev, SergeyMalmö högskola,Fakulteten för hälsa och samhälle (HS)(Swepub:mau)hssesh (författare)
Polaina, JulioInstitute of Agrochemistry and Food Technology, CSIC, Paterna, 46980 Valencia, Spain (författare)
Martinez-Arias, ArturoDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Ferrer, ManuelDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
De Lacey, AntonioDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Fernandez, VictorDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Ballesteros, AntonioDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Plou, FranciscoDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Alcalde, MiguelDepartment of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain (författare)
Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, SpainFakulteten för hälsa och samhälle (HS) (creator_code:org_t)

Ingår i:Combinatorial chemistry & high throughput screening: Bentham Science Publishers Ltd.11:10, s. 807-8161386-20731875-5402

Av författaren/redakt...: Zumarraga, Miren; Dominguez, Crist ...; Camarero, Susana; Shleev, Sergey; Polaina, Julio; Martinez-Arias, ...; visa fler...; Ferrer, Manuel; De Lacey, Antoni ...; Fernandez, Victo ...; Ballesteros, Ant ...; Plou, Francisco; Alcalde, Miguel; visa färre...

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