engleski

Dke1 – Structure, Dynamics and Function, A Theoretical and Experimental Study Elucidated the Role of the Binding Site Shape and the H-Bonding Network in Catalysis

Acetylacetone dioxygenase from Acinetobacter johnsonii (Dke1) utilizes a non-heme Fe2+ center to promote dioxygen-dependent conversion of 2, 4-pentanedione (PD) into methylglyoxal (PR1) and acetate (PR2). In this work we elucidate the role of the protein structure in the catalysis of β-diketone cleavage at the 3-His metal center of Dke1 by computational methods in correlation with kinetic and mutational analyses. Molecular dynamics simulations, using quantum mechanically deduced parameters for the nonheme Fe(II) cofactor, were performed and showed a distinct organization of the hydrophilic triad in the free and substrate ligated wild type enzyme. Our studies show that in the free species, the Fe(II) center is coordinated to 3 histidines and one glutamate, while the substrate ligated, catalytically competent enzyme-substrate complex shows a 3-His Fe(II) center. The substrate binding modes and channels for the traffic of water and ligands (PD, PR1 and PR2) were identified. In order to characterize the impact of the hydrophobic protein environment around the metal center on catalysis, a set of hydrophobic residues lining or close to the active site, namely Phe59, Phe115 and Phe119 were targeted. The variations showed a marked, 20-200 fold impact on the O2 reduction rates. Molecular dynamic studies of the substrate ligated Dke1 variants revealed an impact of the hydrophobic residues on the substrate stabilization in the active site as well as on the orientations of Glu98 and Arg80, which have previously been shown to be crucial for catalysis. Compared to the wild type protein the Glu98-His104 interaction significantly reduced while the interaction between Arg80 and the N terminal from the neighbor unit increased. No correlation between water retention in the active site and O2 reduction rates was observed. Our studies revealed a correlation between the number of low energy positions for O2 in the active site of Dke1 and O2 reduction rates. Based on the obtained results the reaction mechanism is proposed.

Dke1; Fe2+ parameters in nonheme enzyme; Molecular dynamics; Mutants; Random acceleration molecular dynamics

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