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

• Published in: Molecular biology (New York) Vol. 56; no. 4; pp. 600 - 609
• Main Authors: Arteaga-Castrejón, A. A., Trejo-Hernández, M. R., Mekmouche, Y., Amouric, A., Rousselot-Pailley, P., Robert, V., Tron, T., Martínez-Morales, F.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01.08.2022; Springer Nature B.V; MAIK Nauka/Interperiodica
• Subjects: Biochemistry; Biochemistry, Molecular Biology; Biomedical and Life Sciences; Catalysts; Enzymatic activity; Enzymes; Guaiacol; Human Genetics; Hydrophobicity; Immobilization; Laccase; Life Sciences; Lysine; Mutants; pH effects; Site-directed mutagenesis; Structural–Functional Analysis of Biopolymers and Their Complexes; Substrate specificity; Sulfonic acid; site-directed mutagenesis; oriented functionalization; UNIK; laccase
• ISSN: 0026-8933; 1608-3245
• DOI: 10.1134/S0026893322040021

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 (UNIK n , 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 ( and ) determined with ABTS were found to be different among the variants; the was 1.5‒2 fold higher in UNIK 269 and triple mutant, with a of 0.27 and 0.30, respectively; was 30.25 in UNIK 238 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.