On the reaction mechanism of an endoperoxide ring formation by fumitremorgin B endoperoxidase. The right arrangement makes a difference.

Fumitremorgin B endoperoxidase (FtmF) belongs to 2-oxoglutarate dependent dioxygenases and catalyzes an unusual oxidative reaction of endoperoxide formation at the final stage of biosynthesis of verruculogen - a mycotoxin produced by Aspergillus and Penicillinum strains. The published crystal structure of FtmF (PDB: ), which is of overall good quality, contains a model of the substrate bound in the active site, which, however, has very low occupancy and its conformation does not comply with the small molecule crystal structure. Moreover, a previous computational study that employed a model based on this crystal structure revealed a substantial reaction barrier, which might indicate that the model of FtmF/substrate complex can have serious errors. The purpose of this work was to model with computational methods the structure of the enzyme-substrate complex and to investigate the mechanisms of the enzymatic reaction. Docking, molecular dynamics simulation and DFT results, all indicate the substrate most likely binds in the active site in a configuration very different from that originally suggested. Moreover, for this newly proposed structure of the enzyme-substrate complex, the reaction energy profile is characterised exclusively by low barriers and it successfully explains the observed regiospecificity of the enzymatic process. Finally, a plausible binding site for ascorbate was found and it is suggested that ascorbate is involved in the final step of the FtmF reaction.