engleski

Step Forward in Understanding Mechanism of Non-heme Fe2+ Dependent Dioxyhenase Dke1 Catalysis

Acetylacetone dioxygenase from Acinetobacter johnsonii (Dke1) utilizes a non-heme Fe2+ cofactor to promote dioxygen-dependent conversion of 2, 4-pentanedione (PD) into methylglyoxal and acetate. However, the detailed mechanism of the reaction is still unknown. Previous experimental studies assumed that the presence of water molecules in the active site hinders chemical reactions by binding to the metal cation Fe2+ and by shifting the equilibrium of the metal center from the catalytically competent 5-coordinate form to an inept, water protected 6-coordinate species. In order to elucidate the possible role of water molecules in the reaction mechanism, several bulky hydrophobic residues, supposed to be important for the water entrance, were mutated to Ala and the kinetic analysis was performed. Parallel to this, we prepared selected mutants in silico and conducted a detailed computational study as well. We derived parameters for the non-heme ion coordinated by three histidines and either substrate or water molecules, and subsequently in order to track possible differences in the active site architecture and dynamics, we performed a set of about 20 ns molecular dynamics simulations for the wild type protein and the mutants. We determined possible substrate binding modes, water channels, as well as paths for the substrate and the products egress. Using the MM_PBSA approach, we calculated the binding free energy differences for the substrate binding to the wild type protein and the mutants. The computational results show that the selected mutations increased the flexibility of the central part of the protein (region from 60th- 125th residue) and suggest that in order to achieve the full enzymatic effectiveness of Dke1, Arg80, Glu98, and His104 should be in such an orientation that they can tightly interact. Notably, no correlation between the enzyme activity and water retention within the active site was found. Furthermore, we located the most favorable positions for the dioxygen molecule within the active site of the substrate ligated wild type and mutated proteins. The largest number of low energy positions was determined for the wild type enzyme and the lowest for the F115A variant, which was the one with the lowest catalytic activity found in this study.

diketone cleaving enzyme ; Fe2+

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