Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

Abstract

Ethylene is an important signaling molecule in plants that triggers the growth of leaves, flowers, and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate into three CO2 molecules and ethylene. As the detailed mechanism of EFE in the biosynthesis of ethylene remains controversial and particularly the function of the co-substrate l-arginine, we decided to pursue a density functional theory study on the possible pathways of the enzyme leading to ethylene biosynthesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created, which contains all the features of the first- and second-coordination sphere of the active site and substrate (α-ketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either react with a molecule of CO2 to form a carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both the bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form a carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than the typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of the l-arginine co-substrate into the model leads to minor changes in the structure and fold, but its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, the key activation barriers seem less affected by the inclusion of l-arginine into the model. We, therefore, believe that the role of l-arginine is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO2. Our studies show that due to the distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO2 on a persuccinate complex to form carbonate products, leading to ethylene formation. This work gives suggestions on the engineering of EFE into a hydroxylase or improving the ethylene biosynthesis.