Influence of mutations at different distances from the active center on the activity and stability of laccase 13B22

Abstract

Laccases with high catalytic efficiency and high thermostability can drive a broader application scope. However, the structural distribution of key amino acids capable of significantly influencing the performance of laccases has not been explored in depth. Thirty laccase 13B22 mutants with changes in amino acids at distances of 5 Å (first shell), 5-8 Å (second shell), and 8-12 Å (third shell) from the active center were validated experimentally with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. Twelve of these mutants (first shell, 1; second shell, 4; third shell, 7) showed higher catalytic efficiency than the wild-type enzyme. Mutants D511E and I88L-D511E showed 5.36- and 10.58-fold increases in k_cat/K_m, respectively, with increases in optimal temperature of 15 °C and optimal pH from 7.0 to 8.0. Furthermore, both mutants exhibited greater thermostability compared to the wild-type, with increases of 3.33 °C and 5.06 °C in T_m and decreases of 0.39 J and 0.59 J in total structure energy, respectively. The D511E mutation resides in the third shell, while I88L is in the second shell, and their performance enhancements were attributed to alterations in the rigidity or flexibility of specific protein structural domains. Both mutants showed enhanced degradation efficiency for benzo[a]pyrene and zearalenone. These findings highlight the importance of the residues located far from the active center in the function of laccase (second shell and third shell), suggesting broader implications for enzyme optimization and biotechnological applications.

Keywords: Catalytic activity; Laccase; Mutant; Structural distance; Thermostability.

Fig. 1

(A) The 3D structure and Cu ions of 13B22. Cu atoms are marked in red, and 131 candidate residues with distances to the three Cu ions of 13B22 < 12Å are shown in different colors in the three shells. (B) Mutability landscape of 131 candidate residues. The colors of the dots represent the shell. The size of the dots represents the distance to the Cu ions. (C) The position-specific amino acid probability (PSAP) of 28 residues. The rows (A to Y) represent one of the 20 amino acids. Each columns represents one position of 28 residues. The colors from blue to red reflect low to high probability at the evolutionary position, respectively. (D) Distribution of the 28 mutation sites in the structure

Fig. 2

(A) Measured enzymatic activity of single-site mutants and WT 13B22. (B) Measured enzyme activity of multisite combination mutants and WT 13B22

Fig. 3

Effects of pH and temperature on the activity and stability of 13B22, D511E, and I88L-D511E. Optimal (A) temperature and (B) pH for 13B22 and its mutants. (C and D) Temperature and (E and F) pH stability of 13B22 and its mutants

Fig. 4

Protein molecular docking, RMSD, and RMSF analyses of molecular dynamics (MD) simulations. (A) RMSD and (B) RMSF values of the three proteins were calculated by MD simulation using Visual Molecular Dynamics. (C) Structural analysis of 13B22. The active site and the secondary structure with increased or decreased fluctuations are indicated. (D) Spatial relationship between mutated sites 88 and 511 and the active site

Fig. 5

(A) Relative degradation of ZEN catalyzed by WT 13B22, D511E, and I88L-D511E. (B) HPLC analysis of ZEN degradation with WT 13B22, D511E, and I88L-D511E for 1 h. (C) Relative degradation of BaP catalyzed by WT 13B22, D511E, and I88L-D511E. (D) HPLC analysis of BaP degradation with WT 13B22, D511E, and I88L-D511E for 2 h