NATIVE AND COMPLEX CRYSTAL STRUCTURES OF THE FLAVIN ENZYME DIHYDROOROTATE DEHYDROGENASE. Paul Rowland, Finn S. Nielsen, Kaj Frank Jensen & Sine Larsen. Centre for Crystallographic Studies and Center for Enzyme Research, University of Copenhagen, Denmark

High resolution crystal structures of Lactococcus lactis dihydroorotate dehydrogenase "A" have been determined in the native form and also as a complex with the product of the enzyme reaction, orotate. Dihydroorotate dehydrogenase (DOD) is an FMN containing enzyme catalysing the oxidation of L-dihydroorotate to orotate. Lactococcus lactis is the only organism known to contain two functional dihydroorotate dehydrogenases (the "A" and "B" forms). Both consist of polypeptides of 311 residues, though they share only 30% sequence identity. The A form is active as a dimer, whereas the catalytic function of the B form depends strongly on the presence of an iron-sulphur containing protein of 262 amino acids. Well diffracting crystals have been obtained of the A form, which belong to space group P2₁ and have a dimer in the asymmetric unit. The native DODA structure was solved by isomorphous replacement using multiple datasets of a single gold cyanide derivative. Data to 2.0Å has been refined to an R-factor of 16.8% (free R=21.2%). The DODA/orotate complex structure was obtained by soaking a native crystal with the enzyme substrate and has a final R of 16.1% to 2.0Å (free R=18.7%). This first example of a dihydroorotate dehydrogenase structure folds into a classical [[alpha]]/[[beta]] barrel. The flavin binding site is between the top of the barrel and a small [[beta]]-strand sub-domain formed by two barrel inserts. A small cavity above the flavin ring system is completely enclosed by the surrounding protein. In the complex this space is taken up by the orotate without any changes in the protein structure. Many of the conserved residues among this class of enzymes are associated with interactions between the protein residues and the flavin and substrate groups. There are also some important differences between the A and B enzyme forms in nonconserved binding residues.

The DODB/Fe-S protein complex crystallises in space group R32 with one protein complex per asymmetric unit. The results from the investigations of this enzyme will also be presented.