engleski

Diketone cleaving dioxygenase - theoretical study

Diketone cleaving dioxygenase (Dke1) is a non heme Fe(II) enzyme (NHEE) with an atypically ligated metal binding site. Dke1 catalyzes the oxidative C-C bond scission in beta-dicarbonyl compounds by molecular oxygen. The enzyme employs the general mechanism of O2 activation in NHEEs: upon substrate ligation to the iron cofactor, one site of the six-coordinate metal center vacates and O2 is reduced, whereby O2 reduction is the rate determining step of the catalytic cycle. Mutational and spectroscopic analyses have shown that the H-bonding network in the active site plays a crucial role in the rate of O2 reduction. In order to elucidate the structural basis of this effect, the active site was subjected to further mutational analysis. Resulting variants were characterized regarding their O2 reduction rates, and molecular dynamic studies on Dke1 and its mutants were performed, in order to correlate structural data with experimental results. Standard force-field parameters in molecular mechanic programs typically fail to account for the geometric rigidity of NHEE metal binding sites that is found experimentally. As no applicable force field parameters for non-heme Fe(II) sites were available, firstly, the respective force field parameters had to be deduced using quantum mechanics calculations with the program GAMESS. These were incorporated into the AMBER 10 suite applying the AMBER ff03 force field for proteinogenic residues and the general Amber force field for organic molecules. Then this methodology was applied on acetylacetonate ligated Dke1 and selected variants (F59A, F115A, F119A and Y70A) using Molecular Dynamics (MD) simulations (10 ns) and the water entrance into the enzyme active site was monitored. Thus we could identify the primary small channel for water trafficking in the structure of Dke1 and demonstrate that it is distinct from the larger, hydrophobic substrate entrance channel. Furthermore, the structural impact of mutations in the active site on the active site’s accessibility for water was investigated.

binding modes; ab initio calculations; enzyme; MD simulation

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