Simulation of the substrate cavity dynamics of quercetinase.

Molecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu-containing dioxygenase known so far. It catalyses the breakage of the O-heterocycle of flavonols. The substrates occupy a shallow and overall hydrophobic cavity proximal to the metal centre of the homo-dimeric enzyme. The linker connecting the C-terminal and N-terminal domains in the monomer is partly disordered in the crystal structure and part of it forms a flexible lid at the entrance of the substrate cavity. This loop has been tentatively assigned a role in the enzyme mechanism: it helps lock the substrate into place. The dynamics of this loop has been investigated by MD simulation. The initial coordinates were taken from the crystal structure of 2,3QD in the presence of the substrate kaempferol (KMP). After equilibration and simulation over 7.2ns the substrate was removed and another equilibration and simulation of 7.2ns was performed. The results show that the structures of the free enzyme as well as of the enzyme-substrate complex are stable in MD simulation. The linker shows strongly enhanced mobility in the loop region that is close to the entrance to the substrate cavity (residues 154-169). Movement of the loop takes place on a timescale of 5-10ns. To confirm the conclusions about the loop dynamics drawn from the 7.2ns simulation, the simulation was extended with another 8ns. When substrate binds into the cavity the loop orders remarkably, although mobility is retained by residues 155-158. Some regions of the loop (residues 154-160 and 164-176) move over a considerable distance and approach the substrate closely, reinforcing the idea that they lock the substrate in the substrate cavity. The enthalpic component of the interaction of the loop with the protein and the KMP appears to favour the locking of the substrate. Two water molecules were found immobilised in the cavity, one of which exhibited rotation on the picosecond timescale. When the substrate is removed, the empty cavity fills up with water within 200ps.