Atomistic Insights into the Catalytic Mechanism of Propylene Formation by the Non-Heme Fe(II)/2-Oxoglutarate Dependent Ethylene-Forming Enzyme

The Ethylene-forming enzyme (EFE) is a non-heme Fe(II)/2-oxoglutarate (2OG)-dependent oxygenase that uniquely catalyzes the formation of ethylene and 3-hydroxypropionate (3HP) in addition to the hydroxylation of the L-arginine substrate. Recent experimental studies of EFE have demonstrated its ability to utilize 2OG analogs as co-substrates, which, in combination with second coordination sphere (SCS) substitutions, lead to the formation of higher alkenes and/or 3HP derivatives. However, these unique experimental findings have not been explained by substantial mechanistic studies. Gaining mechanistic insight into catalysis by wild-type (WT) and variant EFEs with these 2OG analogs is crucial for understanding the enzyme functions, as well as for facilitating the biosynthesis of valuable organic compounds with industrial relevance. In this study, we implemented molecular dynamics (MD)-based quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the catalytic mechanism of EFE and its L206V variant in complex with 2OG and (4R)-methyl-2OG (4-Me-2OG), which have been experimentally discovered and explored. The study explains the mechanism of how the synergy between the bulk-reducing L206V mutation with the substituted 2OG analog, 4-Me-2OG, enables the formation of propylene. Our results suggest that the L206V variant with 2OG retains key SCS interactions with the active site of the variant, thereby facilitating a similar catalytic mechanism as that of the WT EFE with 2OG, in which the propion-3-yl radical drives ethylene formation and the (2-carboxyethyl)carbonato-Fe(II) (EF-IV) intermediate leads to 3HP production. Our studies of the WT EFE and its L206V variant complexed with 4-Me-2OG indicate that the alternate steric accommodation of the 4-methyl substituent of 2OG within the active site defines the formation of propylene/2-Me-3HP. In the WT EFE, the substituted methyl group of 2OG is oriented away from L206, thus minimizing steric interactions. In contrast, in the L206V variant, the methyl group is directed toward V206, adopting an orientation that closely resembles that of the 2OG in WT EFE. In the L206V variant, the specific orientation and stability of the 2-methyl-propion-3-yl radical are responsible for the formation of propylene and 2-Me-3HP. The calculations of the reaction path starting from the EF-IV intermediate predict that formation 3HP/2-Me-3HP could proceed through an Fe(II)-bound water molecule, similarly to that observed in the WT-EFE with 2OG. Overall, the study underlines the critical role of the synergy between the enzyme's SCS and co-substrate analogs in controlling the divergent reaction pathways for EFE. The present study provides mechanistic insights that might guide the rational redesign of EFE for the biosynthesis of a wide range of alkenes.