Structure-based insights into mechanism of endoperoxidase FtmOx1 catalyzed reactions

FtmOx1 is an alpha-ketoglutarate (alpha-KG) dependent mononuclear non-haem iron enzyme, which catalyzes endoperoxidation in fumitremorgin B to form verruculogen. The main products of the reaction are extremely complicated depending on the different conditions. Two mechanisms, COX-like and CarC-like, were reported, mainly debating which residue Y224 or Y68 is a key determinant in ftmOx1 catalytic endoperoxidation. To address this, here, we performed structural analysis of three binary complexes, ftmOx1 center dot Fe2+center dot alpha-KG (4Y5S), ftmOx1 Y68F center dot Co2+center dot alpha-KG (7ETL) and ftmOx1 Y140F center dot Fe2+center dot alpha-KG (6OXH), and of two ternary complexes, ftmOx1 center dot Co2+center dot alpha-KG center dot 13-oxo fumitremorgin B (7WSB) and ftmOx1 center dot Fe2+center dot alpha-KG center dot fumitremorgin B (7ETK). We found that residue Y224 forms a hydrogen bond with alpha-KG, which implies that Y224 potentially suppresses oxygen rebound reaction by forming a hydrogen bond with oxo-ferry species and enhances the endoperoxidation selectivity of wild-type ftmOx1. Substrate insertion into the Fe2+ binding pocket results in the generation of a new conformation of Y224, in line with the observed structures of 7ETK and 7WSB. This conformation of Y224 is fixed by forming a hydrogen bond with the residue T134 -OH group. The mutations of residue T134 led to major products of oxygen rebound and dealkylation, suggesting that it acts as a regulator of the reactions. Possible reaction pathways were suggested, demonstrating how Y68, T134 and Y224 work together in ftmOx1 catalyzed reactions.