Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme.
Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5-9.0. It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2%. Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.
Nielsen
F S
FS
Center for Enzyme Research, University of Copenhagen, Denmark.
Rowland
P
P
Larsen
S
S
Jensen
K F
KF
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D000090722:Dihydroorotate Dehydrogenase; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D000090722:Dihydroorotate Dehydrogenase; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors