Methylene Group Insertion into a C–N Bond: The Mechanism for the Biosynthesis of Dehydrofosmidomycin by a Nonheme Iron Oxygenase

The nonheme iron dioxygenase dehydrofosmidomycin synthase D (DfmD) performs an unusual catalytic reaction mechanism with an initial desaturation followed by N-demethylation and the insertion of a CH₂ group into a C–N bond. However, the reaction process is challenging synthetically and an enzymatic approach by a single protein may have applications in biotechnology for the biosynthesis of drug molecules. Little is known regarding the details of the reaction mechanism and several possible mechanisms have been proposed. To resolve the controversy, we performed a detailed computational study on the desaturation and rearrangement steps of 2-(trimethylammonio)ethylphosphonate by DfmD using two molecules of O₂ and α-ketoglutarate to form methyldehydrofosmidomycin. Interestingly, 2-(trimethylammonio)ethylphosphonate dioxygenase (TmpA) activates the same substrate but produces C₁-hydroxylation products instead and only runs one catalytic cycle with the substrate. Our computational study uses molecular dynamics and quantum mechanics calculations and focuses on the comparison of the structure, mechanism, and function of the two enzymes. The initial molecular dynamics simulation shows that the substrate is tightly bound to the substrate-binding pocket although its position differs from that in TmpA. We created enzymatic models and studied the substrate-binding and reaction mechanisms leading to products and potential byproducts for the two successive catalytic cycles with an iron(IV)-oxo species. Molecular dynamics simulations confirm that the product after the first cycle does not escape the protein in DfmD. Our calculations confirm experimental product distributions and show a dominant C₁-hydroxylation mechanism for TmpA, whereas DfmD reacts through a dominant desaturation of the C₁–C₂ bond with two consecutive hydrogen atom abstraction steps from the C₁–H and C₂–H bonds. A second oxidation cycle with O₂ and α-ketoglutarate starts with hydrogen atom abstraction from the N-methyl group followed by CH₂ insertion into the C–N bond and OH rebound to form methyldehydrofosmidomycin products. We analyzed the optimized geometries, charge distributions, and substrate mobility patterns and concluded that the active site of TmpA binds the substrate strongly through salt bridges and hydrogen bonding interactions that guide the selectivity to C₁-hydroxylation, while the different polarity and local electric field directions of the binding pocket in DfmD guide the reaction to desaturation instead.