Revealing the nature of the second branch point in the catalytic mechanism of the Fe(ii)/2OG-dependent ethylene forming enzyme.

Ethylene-forming enzyme (EFE) has economic importance due to its ability to catalyze the formation of ethylene and 3-hydroxypropionate (3HP). Understanding the catalytic mechanism of EFE is essential for optimizing the biological production of these important industrial chemicals. In this study, we implemented molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) to elucidate the pathways leading to ethylene and 3HP formation. Our results suggest that ethylene formation occurs from the propion-3-yl radical intermediate rather than the (2-carboxyethyl)carbonato-Fe(ii) (EFIV) intermediate, which conclusively acts as a precursor for 3HP formation. The results also explain the role of the hydrophobic environment surrounding the 2OG binding site in stabilizing the propion-3-yl radical, which defines their conversion to either ethylene or 3HP. Our simulations on the A198L EFE variant, which produces more 3HP than wild-type (WT) EFE based on experimental observations, predict that the formation of the EFIV intermediate was more favored than WT. Also, MD simulations on the EFIV intermediate in both WT and A198L EFE predicted that the water molecules approach the Fe center, which suggests the role of water molecules in the breakdown of the EFIV intermediate. QM/MM simulations on the EFIV intermediate of WT and A198L EFE predicted that the Fe-bound water molecule could provide a proton for the 3HP formation from EFIV. The study underscores the critical influence of the enzyme's hydrophobic environment and second coordination sphere residues in determining product distribution between ethylene and 3HP. These mechanistic insights lay a foundation for targeted enzyme engineering, aiming to improve the selectivity and catalytic efficiency of EFE in biological ethylene and 3HP production.