Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase - Aix-Marseille Université Access content directly
Journal Articles Molecular Biology Year : 2022

Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase

Pertinence des résidus de lysine de surface pour la fonctionnalisation de Laccase

Abstract

Fungal laccases are oxidoreductases with low-specificity for substrates. The characterization of laccase’s surface is a prerequisite used to obtain hybrid catalysts with new properties. Surface-exposed lysine residues are targets in immobilization reactions. In this work, LAC3-K0, an enzyme devoid of lysine, was used as a platform to detect potential surface-exposed sites suitable for replacement with a lysine residue. Seven sites were selected from a LAC3-K0 3-D model, and single lysine mutants (UNIKn, n = residue number) were obtained by site-directed mutagenesis. All mutants were expressed in Saccharomyces cerevisiae W303-1A and detected as functional secreted proteins by their ability to oxidize guaiacol or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) on agar plates. All variants were active at acidic pH but presented no activity at neutral pH, as expected. Likewise, variants were stable a temperature between 15‒55°C, and were completely inactivated at 70°C. Oxidation assays revealed that the replacement of one or two surface residues with lysine greatly affected enzyme activity and substrate specificity. The catalytic parameters (KappM and kappcat) determined with ABTS were found to be different among the variants; the Vappmax was 1.5‒2 fold higher in UNIK269 and triple mutant, with a KappM of 0.27 and 0.30, respectively; kappcat was 30.25 in UNIK238 and 32.34 in the triple mutant. The role of hydrophobic patches detected on the surface of LAC3-K0 was determined to be a favorable factor to be considered in the interaction of hybrid materials. All variants with uniquely surface located lysine created in this work can be in demand for obtaining laccases with a certain substrate specificity in the design of hybrid materials.
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Dates and versions

hal-03842442 , version 1 (26-01-2023)

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A. Arteaga-Castrejón, M. Trejo-Hernández, Yasmina Mekmouche, A. Amouric, P. Rousselot-Pailley, et al.. Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase. Molecular Biology, 2022, 56 (4), pp.600-609. ⟨10.1134/S0026893322040021⟩. ⟨hal-03842442⟩
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