Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyze the Mycotoxin Zearalenone

Abstract

Zearalenone (ZEN) is a mycoestrogenic polyketide produced by <i>Fusarium graminearum</i> and other phytopathogenic members of the genus <i>Fusarium</i>. Contamination of cereals with ZEN is frequent, and hydrolytic detoxification with fungal lactonases has been explored. Here, we report the isolation of a bacterial strain, <i>Rhodococcus erythropolis</i> PFA D8-1, with ZEN hydrolyzing activity, cloning of the gene encoding α/β hydrolase ZenA encoded on the linear megaplasmid pSFRL1, and biochemical characterization of nine homologues. Furthermore, we report site-directed mutagenesis as well as structural analysis of the dimeric ZenA_Re of <i>R. erythropolis</i> and the more thermostable, tetrameric ZenA_Scfl of <i>Streptomyces coelicoflavus</i> with and without bound ligands. The X-ray crystal structures not only revealed canonical features of α/β hydrolases with a cap domain including a Ser-His-Asp catalytic triad but also unusual features including an uncommon oxyanion hole motif and a peripheral, short antiparallel β-sheet involved in tetramer interactions. Presteady-state kinetic analyses for ZenA_Re and ZenA_Scfl identified balanced rate-limiting steps of the reaction cycle, which can change depending on temperature. Some new bacterial ZEN lactonases have lower <i>K</i>_M and higher <i>k</i>_cat than the known fungal ZEN lactonases and may lend themselves to enzyme technology development for the degradation of ZEN in feed or food.