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 dihydroor otate dehydrogenases, i.e., the A- and B-forms. In this paper, we repo rt the overproduction, purification, and crystallization of dihydrooro tate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin monon ucleotide 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 dich lorophenolindophenol, potassium hexacyanoferrate(III), and, to a lower extent, also molecular oxygen as accepters of the reducing equivalent s, whereas the pyridine nucleotide coenzymes (NAD(+), NADP(+)) and the respiratory quinones (i.e., vitamins Q(6), Q(10) and K-2) were inacti ve. The enzyme has been crystallized from solutions of 30% polyethylen e glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The result ing yellow crystals diffracted well and showed little sign of radiatio n damage during diffraction experiments. The crystals are monoclinic, space group P2(1) with unit cell dimensions a = 54.19 Angstrom, b = 10 9.23 Angstrom, c = 67.17 Angstrom, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 Angstr om and an R(sym) value of 5.2%. Analysis of the solvent content and th e self-rotation function indicates that the two subunits in the asymme tric unit are related by a noncrystallographic twofold axis perpendicu lar to the crystallographic b and c axes.