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The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers

¹ Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1307, Copenhagen K, Denmark
² Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, DK-2100, Copenhagen 0, Denmark
³ Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606 USA

Reprint requests to: Kaj Frank Jensen, Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK 1307 Copenhagen K, Denmark; e-mail: kfj{at}mermaid.molbio.ku.dk; fax: +45 3532 2040.

The flavoenzyme dihydroorotate dehydrogenase A from Lactococcus lactis is a homodimeric protein of 311 residues/subunit, and the two active sites are positioned at a distance from the dimer interface. To promote formation of the monomeric form of the enzyme, we changed the residues involved in formation of two salt bridges formed between the residues Glu206 of the one polypeptide and Lys296 of the other polypeptide. The mutant enzymes formed inactive precipitates when cells were grown at 37°C, but remained soluble and active when cells were grown at 25°C. The salt bridges were not needed for activity, because the mutant enzymes in which one of the residues was converted to an alanine (E206A or K296A) retained almost full activity. The mutant enzymes in which the charge of one of the residues of the salt bridge was inverted (i.e., E206K or K296E) were severely impaired. The double mutant E206K-K296E, which has the possibility of forming salt bridges in the opposite orientation of the wild type, was fully active in concentrated solutions, but dissociated into inactive monomers upon dilution. The K_D for the dimer to monomer dissociation reaction was 12 µM, and dimer formation was favored by the product, orotate, or by high ionic strength, indicating that the hydrophobic interactions are important for the subunit contacts. Wild-type dihydroorotate dehydrogenase A was similarly found to dissociate into inactive monomers, but with a K_D for dissociation equal to 0.12 µM. These results imply that the dimeric state is necessary for activity of the enzyme.

Keywords: Nucleotide synthesis; oligomerization; quaternary structure; dissociation kinetics; oxido-reductase; flavin

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