Experimental-theoretical study of laccase as a detoxifier of aflatoxins

Abstract

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB₁ and AFG₂) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB₁ is more challenging to degrade, to the point of complete degradation stalling; (2) AFG₂ is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Figure 1

The detoxification of AFB₁ and AFG₂ by laccase highlights the difference in detoxification efficiencies even between aflatoxins with similar structure. Different initial aflatoxin concentrations were employed and are represented for AFB₁ (A) and AFG₂ (B). Each curve is the average of 3 replicates. A subset of points from (A,B) is randomly selected and represented in (C,D) to calculate the local normalized detoxification rates $\left(\frac{-1}{\left[L\right]},\frac{d\left[T\right]}{\mathit{dt}}\right)$. Here, $\left[T\right]$ is the toxin concentration and $\left[L\right]$ is the laccase concentration. Detoxification efficiency $\left(\eta ,\stackrel{\mathrm{def}}{=},\frac{-1}{\left[L\right]\left[T\right]},\frac{\mathrm{\Delta }\left[T\right]}{\mathrm{\Delta }t}\right)$ of AFB₁ (C) is almost an order of magnitude lower than that of and AFG₂ (D) at comparable concentrations. Dotted lines in (C,D) illustrate the prediction of the model. Direction of time is represented in (C,D) to highlight the decrease in toxin concentration as a result of detoxification. Laccase concentration: 25 U/mL.

Figure 2

Isosurfaces of the Fukui functions of AFB₁ and AFG₂ in the gas phase indicate the sites prone to oxidation. Fukui isosurfaces: red (−) and blue (+). Isosurface level of ± 0.003 Å⁻³.

Figure 3

The main reaction products of AFB₁ and AFG₂ break-down, as identified by LC–MS and hypothetical reaction pathways. The main proposed mechanisms are the lactone ring opening and epoxide formation.

Figure 4

Interaction energies of different poses of AFB₁ and AFG₂ extracted from an MD simulation using a cluster model show no major differences between the congeners. Energies are shown for two different QM models (PBE + D3 and B97M-V).

Figure 5

Heat map of interactions between laccase residues and low energy toxin poses as measured by FBO highlights residues of interest. A higher FBO indicates a stronger interaction.