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‐HC1, 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 P2, with unit cell dimensions a = 54.19 Å, b = 109.23 Å, c = 67.17 Å, and β = 104.5°. A native data set has been collected with a completeness of 99.3% to 2.0 Å 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.