Discussion and Path Ahead
Laccase enzymes are a type of multi-copper species, which catalyze the oxidation of substrate molecules to corresponding reactive radicals with the simultaneous reduction of oxygen to water at a mononuclear copper center type-1(T1). The catalytic center of these enzymes is composed of four copper atoms. Two histidine residues and one cysteine residue create a metallo-organic link with the T1 copper atom. Additionally, the T1 copper is in close proximity to the side chains of a methionine, leucine, or isoleucine. Two histidine ligands coordinate the trinuclear center's type 2 (T2) copper atom, whereas a total of six histidine ligands coordinate the two types 3 (T3) copper atoms.
The enzyme can oxidize a variety of substances, including polyphenols, methoxy substituted phenols, aromatic diamines, and others. Laccase enzymes are used in a variety of biotechnological processes, such as the delignification of lignocellulosic materials, bio-pulping and bio-bleaching, transformation of textile dyes, removal of phenolics from must and wine, and detoxification of effluent containing phenolic waste. An explicit description of the molecular determinants of their substrate specificity are yet to be deciphered. Considerable number of crystal structures of fungal laccases have already been determined, hence providing us with a diversity of templates for further study of the molecular interaction between the enzyme and its substrates.
The affinity of laccase toward AFB1 and AFG1 depends on the ability of the enzyme to form hydrogen bonds, ionic interactions, water bridges and hydrophobic contacts with the two toxin variants of Aflatoxin. In the current study, We have performed docking studies of the wild-type (WT) laccases to establish base levels of binding affinity of AFB1 and AFG1, while simultaneously trying to establish if laccases possess the affinity to bind and subsequently degrade different variants of Aflatoxins. Favorable mutations were found using critical interacting residues in the WT molecular docking, using CUPSAT server and HotSpot Wizard. Twenty favorable and stabilizing point mutations found were individually introduced to the laccase enzyme and the impact was tested using molecular docking.
From the results generated, 4 best results were filtered and their stacking effect was tested using a multiple mutation approach. The results were not as beneficial as individual mutations. Only two best point mutations were selected namely 106_asp-lys and 181_asp-trp. These were further tested using Molecular Dynamics approach to prove stabilities of their protein-ligand complexes in a water-based system and simultaneously validate our molecular docking results. The 181_asp-trp_afb complex, when compared to the wt-laccase_afb1 complex, was found to possess stronger protein-ligand interactions, and stable structural conformations deduced from reduced RMSF and RMSD values. 106_asp-lys_afg1 complex when compared to wt-laccase-afg1 complex showed an improved protein-ligand interaction despite having a slight increase in protein RMSD values.
All experimentation was conducted in a dry lab and further requires wet-lab testing to fortify our findings.
The applications of the laccase degradation activity lies towards having safer food work and also works under the Goal 2 -> 0 Hunger and Goal 3 -> Good Health and Well Being of the Sustainable Development Goals.
