Escherichia coli dihydroorotase has been crystallized in the presence of the product, L-dihydroorotate (L-DHO), and the structure refined at 1.9 angstrom resolution. The structure confirms that previously reported (PDB entry 1]79), 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-aspbound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the LDHO-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. (c) 2005 Elsevier Ltd. All rights reserved.