Study on DepA preparation by cell free expression system and elucidation of DON biotransformation mechanisms by...

Deoxynivalenol (DON) is a mycotoxin widely present in grain crops and feed materials and exhibits a relatively high contamination rate. It is most commonly found in grains such as wheat, barley, and corn. DepA, a pyrroloquinoline-quinone (PQQ)-dependent enzyme isolated from Devosia mutans 17-2-E-8, catalyzes the conversion of DON to 3-keto-DON. Following the synthesis and cloning of the DepA gene, the deoxynivalenol epimerization enzyme (DepA) was produced using a cell-free Escherichia coli synthesis system. DepA has a molecular weight of 63 kDa, with optimal activity at pH 7.0 and 35 °C. The coenzyme PQQ is essential for the activity of both crude and purified DepA. Metal ions Ca²⁺ enhance DepA activity, while Co²⁺,Cu²⁺ and Zn²⁺ inhibit. Three-dimensional structural simulation of the DepA protein (covering 90.88 % of the gene sequence) identified a PQQ binding site and a Ca²⁺ binding site. This indicates that PQQ influences the binding of DON to DepA. In the presence of the coenzyme PQQ, the type and strength of interactions between PQQ and DON changed: the interaction distance with the hydrogen atom of the C3-OH group decreased, while the interaction with the hydrogen atom at the C3 position increased. This alteration may be an important factor underlying PQQ's influence on DepA activity. This study provides a novel method for producing DepA using a cell-free system and elucidates a mechanistic pathway for DepA-catalyzed biotransformation of DON into 3-keto-DON.