Mechanistic Insight from Full Quantum Mechanical Modeling: Laccase as a Detoxifier of Aflatoxins

Abstract

We demonstrate a path towards full Quantum Mechanics (QM) characterization of enzymatic activity. As a case-study, we investigate the detoxification of aflatoxin, a carcinogenic food contaminant, by laccase, a versatile oxidase capable of—but not efficient for—degrading aflatoxin. We use a combination of quantitative experimentation and QM modeling to show that low enzymatic steric affinity for aflatoxin is the main bottleneck, rather that the oxidative activity of laccase. To identify the structural elements responsible for low reaction rates, we perform a density functional theory (DFT) based modeling of both the substrate and the enzyme in a full QM simulation of more than 7,000 atoms. Thanks to our approach we point to amino acid residues that determine the affinity of laccase for aflatoxin. We show that these residues are substrate-dependent, making a full QM approach necessary for enzyme optimization. Altogether, we establish a roadmap for rational enzyme engineering applicable beyond our case study.

Partner-assisted artificial selection of a secondary function for efficient bioremediation

“…Additionally, PAAS offers the advantage that growth as the primary trait of interest is relatively easy to measure, compared to direct measurements of the toxin concentration, e.g. through fluorescence [13], ELISA, or HPLC [14,15]. shows a positive correlation between the detoxification rate and total cell density.…”

Enzyme fatigue limits the detoxification of aflatoxin by Rhodococcus species

“…The readouts are also normalized to fluorescence data from no enzyme controls to remove the effect of fluorescence loss due to bleaching or other causes over time. The resulting values are then transformed to toxin concentration using a calibration data that contained values corresponding to known enzyme concentrations (34).…”

Computational biology for enzymatic removal of mycotoxins

“…Isolates can be cultivated and tested for degradation, especially when high-throughput screening is possible. For example, AF was shown to be degraded by Rhodococcus erythropolis via an excreted enzyme, and additionally AF presence can be detected via the fluorescence of its lactone ring, which is broken during degradation (52). Such a system can be easily assayed by fluorescent assays of the supernatant.…”

“…An example of this is calculating a Fukui function of a molecule, which describes the change in a frontier orbital as the molecule undergoes a redox reaction. Fukui functions have been used to identify the location of redox in an AF-laccase system (52). QM may also be used to study electron transfer in the protein.…”