Dke1—structure, dynamics, and function: a theoretical and experimental study elucidating the role of the binding site shape and the hydrogen-bonding network in catalysis

Abstract

This study elucidates the role of the protein structure in the catalysis of β-diketone cleavage at the three-histidine metal center of diketone cleaving enzyme (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. It is shown that in the free species, the Fe(II) center is coordinated to three histidines and one glutamate, whereas the substrate-ligated, catalytically competent enzyme-substrate complex has an Fe(II) center with three-histidine coordination, with a small fraction of three-histidine, one-glutamate coordination. The substrate binding modes and channels for the traffic of water and ligands (2,4-pentandionyl anion, methylglyoxal, and acetate) were identified. To characterize the impact of the hydrophobic protein environment around the metal center on catalysis, a set of hydrophobic residues close to the active site were targeted. The variations resulted in an up to tenfold decrease of the O(2) reduction rates for the mutants. Molecular dynamics studies 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. Consequently, the Glu98-His104 interaction in the variants is weaker than in the wild-type complex. The role of protein structure in stabilizing the primary O(2) reduction step in Dke1 is discussed on the basis of our results.

Insight of the Iron Binding and Transport in Dke1 - A Molecular Dynamics Study

“…Systems were parameterized using the AMBER [9] ff10 [10] force field, GAFF [11] (General Amber force field), and the parameters for Fe 2+ ion that were derived and tested earlier. [5,12] Parameters development was achieved with ab initio quantum mechanical calculations (using UHF method). [5] Equilibrium values for the bond distances, angles, and dihedrals, were determined, as well as their corresponding force constants.…”

“…[5,12] Parameters development was achieved with ab initio quantum mechanical calculations (using UHF method). [5] Equilibrium values for the bond distances, angles, and dihedrals, were determined, as well as their corresponding force constants. According to the experimental conditions, [2][3][4][5] the all simulations were performed at pH 7.5.…”

“…[5] Equilibrium values for the bond distances, angles, and dihedrals, were determined, as well as their corresponding force constants. According to the experimental conditions, [2][3][4][5] the all simulations were performed at pH 7.5. Histidines were uncharged (singly protonated), aspartic and glutamic acids were negatively, and arginines and lysines positively charged.…”

“…In the crystallographically determined structure the metal ion is coordinated with three histidines, while the molecular dynamics study showed that beside the histidines one glutamate could also take part in the metal ion coordination. [5] Although the Dke1 active site, besides the Fe 2+ ion, can host several other metal ions, such as Co(II), Ni(II), Cu(II) and Zn(II), the enzyme is unable to catalyze the oxidative conversion of acetylacetone in their presence. [4] Until now only the effects of hydrophilic residues, placed in the second coordination sphere of the metal, have been investigated computationally, using the molecular dynamics (MD) simulations.…”

“…[4] Until now only the effects of hydrophilic residues, placed in the second coordination sphere of the metal, have been investigated computationally, using the molecular dynamics (MD) simulations. [5] The main purpose of this study was to get insights into molecular mechanisms of Dke1, which haven't been demystified by now. Using computational methods the specific and non-specific metal ion binding sites are revealed and amino acid residues responsible for metal transport in and out of the Dke1 active site are identified.…”