Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits

Escherichia coli dihydroorotase has been crystallized in the presence of the product, l-dihydroorotate (l-DHO), and the structure refined at 1.9 Å resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-d,l-aspartate (d, l-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, l-CA-asp bound at the active site and the other having l-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105–115 has different conformations in the two subunits. In the case of the l-CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the l-DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of l-DHO to l-CA-asp at low concentrations of l-DHO shows positive cooperativity with a Hill coefficient n=1.57(±0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.