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 1.9A 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.
Lee
Mihwa
M
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Chan
Camilla W
CW
Mitchell Guss
J
J
Christopherson
Richard I
RI
Maher
Megan J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004080:Dihydroorotase / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D021122:Protein Subunits; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D004080:Dihydroorotase