The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers.
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 degrees C, but remained soluble and active when cells were grown at 25 degrees 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 microM, 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 microM. These results imply that the dimeric state is necessary for activity of the enzyme.
Ottosen
Mette Brimheim
MB
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1307, Copenhagen K, Denmark.
Björnberg
Olof
O
Nørager
Sofie
S
Larsen
Sine
S
Palfey
Bruce Allan
BA
Jensen
Kaj Frank
KF
D000090722:Dihydroorotate Dehydrogenase; D019281:Dimerization; D005816:Genetic Complementation Test; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009963:Orotic Acid / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D020836:Protein Structure, Quaternary
D000090722:Dihydroorotate Dehydrogenase; D009963:Orotic Acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors