Bioremediation of aflatoxins by DypB: towards a full understanding of the reaction mechanism by time-resolved structural investigations

Peroxidases are heme-containing enzymes catalysing the oxidation of aromatic compounds by hydrogen peroxide. Members of the dye-decolouring subfamily (Dyps) are well known for their activity over a variety of substrates including aflatoxins (toxic fungal metabolites responsible for cereal and nut contaminations). Preliminary data obtained by IC-CNR show that aflatoxin B1 (AFB1) binds the reduced (inactive) form of DypB from Rhodococcus jostii and a crystallographic structure of the protein-substrate complex has been determined. Despite its vicinity to the heme, the substrate position is incompatible with the electron transfer mechanism hypothesized for DypB that includes the formation of radical cation Fe(IV) by H2O2, its conversion to Fe(IV)=O and final reduction to Fe(III). Aim 1: To describe the mechanism of AFB1 oxidation by DypB using time-resolved X-ray crystallography coupled with micro-spectrophotometry. These techniques will allow complementing the structure of the toxin-bound inactive enzyme with other snapshots of the catalytic cycle and characterising the transition between the apo form and the active state, as well as the intermediate states of the reaction.
Aim 2: To couple crystallographic results with molecular docking calculations, to provide insight into key residues involved in each step of the reaction. This will pave the way to the engineering of DypB for bioremediation of cattle feed.

Aim 3: To standardise a crystal crosslinking method yielding stable DypB-based heterogeneous catalysts to be used in different chemical environments and applicable to a wider range of substrates.