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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

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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 O2 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 O2 reduction step in Dke1 is discussed on the basis of our results.

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  1. Structures of an Fe(III) containing quercentin 2,3-dioxygenase from Bacillus subtilis (PDB code 1Y3T) and of Fe(II) ligated acireductone dioxygenase from Klebsiella oxytoca (PDB code 2HJ1).







Molecular dynamics






2,4-Pentanedionyl anion


Protein Data Bank


Random-acceleration molecular dynamics




Unrestricted Hartree–Fock


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This work is the result of the bilateral Croatian–Austrian collaboration project “Metal centers in enzymes and proteins.” The authors acknowledge the Ministry of Science, Education, and Sport of the Republic of Croatia (project 098-0982913-2748) and the Austrian Academic Exchange Service (WTZ project HR 26/2008) for their financial support. G.D.S. acknowledges the support of the FWF (P18828).

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Correspondence to Grit Straganz or Sanja Tomić.

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Brkić, H., Buongiorno, D., Ramek, M. et al. 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. J Biol Inorg Chem 17, 801–815 (2012).

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