In silico Design of Laccase Thermostable Mutants From Lacc 6 of Pleurotus Ostreatus

Abstract

Fungal laccase enzymes have a great biotechnological potential for bioremediation processes due to their ability to degrade compounds such as ρ-diphenol, aminophenols, polyphenols, polyamines, and aryldiamines. These enzymes have activity at different pH and temperature values, however, high temperatures can cause partial or total loss of enzymatic activity, so it is appropriate to do research to modify their secondary and/or tertiary structure to make them more resistant to extreme temperature conditions. In silico, a structure of the Lacc 6 enzyme of Pleurotus ostreatus was constructed using a laccase of Trametes versicolor as a template. From this structure, 16 mutants with possible resistance at high temperature due to ionic interactions, salt bridges and disulfide bonds were also obtained in silico. It was determined that 12 mutants called 4-DB, 3-DB, D233C-T310C, F468P, 3-SB, L132T, N79D, N372D, P203C, P203V, T147E, and W85F, presented the lowest thermodynamic energy. Based on the previous criterion and determining the least flexibility in the protein structures, three mutants (4-DB, 3-DB, and P203C) were selected, which may present high stability at high temperatures without affecting their active site. The obtained results allow the understanding of the molecular base that increase the structural stability of the enzyme Lacc 6 of Pleurotus ostreatus, achieving the in silico generation of mutants, which could have activity at high temperatures.

Keywords: Lacc 6; Pleurotus ostreatus; energy minimization; laccase; mutants.

FIGURE 1

Model of the laccase enzyme of Pleurotus ostreatus. In color-coded the secondary structure is presented: yellow, beta sheets; purple and blue, alpha helix; in green and white, loops and non-structured areas. In ocher color the copper atoms belonging to the active site are represented. The 180 degrees rotation allows to appreciate the three structural domains which are defined by Domain A of amino acids 1–131, Domain B of amino acids 132–301 and Domain C the last 194 amino acids.

FIGURE 2

Propensity diagram for the alignment of the 16 thermoresistant sequences. In blue color the conservation of Histidines that conform the active site for this class of enzyme is shown, the positions of greater conservation (greater amplitude) generate clues of evolutionary invariants and important for the structural stability of these enzymes.

FIGURE 3

Values of mean displacement for the difference between the mutated enzyme and its native counterpart.

FIGURE 4

Simulation performed in ElNemo of low frequency movements for Lacc 6 and mutant 3-DB. An overlap of the 10 snapshots calculated within mode 7 of the mutated enzyme (in colors according to its secondary structure) on the native enzyme (gray scale), the low/high frequency of the structure indicated by the movement of the Cα which suffer a displacement of between 1.00 and 1.20 Å.

FIGURE 5

Structural overlap illustrating Active Site disturbances due to the mutation performed by replacing side chain Lysine 132 with a Threonine (L132T). The color image corresponds to the mutant enzyme and in grayscale to the native enzyme.

FIGURE 6

Low frequency motions corresponding to mode 7 calculated by ElNemo show a counter-rotation torsion between the domain for the distant copper and the binding domains to trinuclear site. In general, the perturbation to the global structure generated by the mutants: 3-SB (A), L132T (B), P203C (C), and F468P (D) is appreciated. ElNemo, overlapping of low energy motions on the minimized conformation between the native enzyme (grayscale) and the mutant (color coding according to secondary structure). These animations circulate around the 36 conformational snapshots, stored for low-frequency mode 7, centered on the minimized conformation of each type of enzyme. The regions of the most frequent proteins are highlighted given the counter-rotation movements between structural domains.

FIGURE 7

Relative movements in ElNemo mode7 between the mutant P203C and the native enzyme. The proteins are represented in the form of slats. The color-coded mutant depending on its secondary structure and native enzyme in grayscale. Also, it is shown an increase in fluctuation of native enzyme and the interconversion of non-random secondary structure for the formation of a beta-sheet on the opposite side to the mutation.

FIGURE 8

Domains close to the copper atom away from the trinuclear site of copper, showing the detail of the splicing of histidines bound to copper atoms.