INTERACTION AND STRUCTURAL FLEXIBILITY OF A Phlebia brevispora BAFC633 LACCASE WITH 2,4-D AND CHLORPYRIFOS

AYALA SCHIMPF AR; FONSECA, MI.; ZAPATA, PD.,

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiologia General SAMIGE; 2023

The widespread use of agrochemicals in agriculture, such as 2,4-dichlorophenoxyacetic acid (2,4-D) and Chlorpyrifos (CP), raises constant environmental concerns. Phlebia brevispora BAFC 633 is a white-rot fungus known for its metabolic capability to degrade complex compounds like pesticides, attributed to enzyme production, including laccase. Laccases (EC 1.10.3.2) belong to multicopper oxidases, catalyzing the oxidation of various phenolic compounds, and actively involved in environmental contaminant degradation. In addition to in vitro studies, bioinformatics research enhances understanding of this intricate interaction, seeking effective biotechnological solutions. Therefore, this work aims to assess laccase stability against 2,4-D and CP, employing molecular dynamics to comprehend their interactions and structural flexibility.Molecular Dynamics (MD) simulations were conducted using NAMD 3.0.0 software with Charmm 27 force field. Receptors were built using NAMD's System Builder and solvated in an octahedral TIP3P solvent box with 0.15 M Na+ and Cl- ions. Thermodynamic stability was assessed through RMSD, RMSF, H-BOND, and SASA. For qualitative analysis of 2,4-D and CP binding, three simulations per system were performed, 1 of 50 ns (runs 1 and 2), and 1 of 250 ns (run 3) for independence assurance. Binding enthalpy contributions were calculated via MM-PBSA from AMBER 18 package. One hundred frames from trajectories were processed, and the net system energy was estimated using the equation ΔGBinding = ΔGComplex – ΔGReceptor – ΔGpesticide.RMSD for the LacI-CP complex maintained its position within a 2 Å range initially, exhibiting increasing mobility without deviating from the active site or surrounding amino acids. This stability persisted across all repetitions. In contrast, the LacI-2,4D complex displayed greater mobility with elevated RMSD values around 100 ns. RMSF revealed enhanced mobility in the 285-305 loop. SASA analysis remained steady, indicating minimal structural changes. Although CP exhibited lower initial SASA, it did not affect its interaction with the protein. Both complexes formed hydrogen bonds with the enzyme, more prominently observed in LacI-CP. CP maintained its initial position throughout the 250 ns, while 2,4-D displayed greater mobility, moving away and forming connections with various receptor residues. MM-PBSA analysis unveiled predominantly polar interactions for CP and 2,4-D, maintaining distinct hydrogen and hydrophobic bonds.This molecular-level exploration will yield significant insights for further enhancing LacI's function, uncovering residue details that can facilitate its biotechnological engineering, modification, and industrial-scale applications.