CRYSTALLIZATION AND PRELIMINARY-X-RAY DIFFRACTION ANALYSIS OF THE HETEROTETRAMERIC DIHYDROOROTATE DEHYDROGENASE-B OF LACTOCOCCUS-LACTIS, A FLAVOPROTEIN ENZYME-SYSTEM CONSISTING OF 2 PYRDB SUBUNITS AND 2 IRON-SULFUR CLUSTER CONTAINING PYRK SUBUNITS

Dihydroorotate dehydrogenases are flavin-containing enzymes which cata lyze the conversion of (S)-dihydroorotate to orotate. Dihydroorotate d ehydrogenase B (DHODB) from Lactococcus lactis is a heterotetramer con taining two subunits of the protein encoded by the pyrDb gene (PyrDB) and two subunits of the protein encoded by the pyrK gene (PyrK). In ad dition, DHODB contains two molecules of flavin mononucleotide, two mol ecules of flavin adenine dinucleotide and two [2Fe-2S] iron-sulfur clu sters as tightly bound cofactors. Yellow crystals of this enzyme have been grown using the hanging-drop vapour diffusion technique from solu tions of 2.5 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6. The crystals have been shown to contain both the PyrDB and the PyrK subuni ts and fluorescence measurements indicate that the two different subun its interact very closely with each other in the active-site region. N ative data sets have been collected to 2.6 Angstrom with a conventiona l X-ray source and to 2.2 Angstrom using synchrotron radiation. The cr ystals are rhombohedral, space group R32, with corresponding hexagonal unit-cell dimensions a = b = 202.3 and c = 81.0 Angstrom. The asymmet ric unit in the crystal contains one PyrDB subunit and one PyrK subuni t, which suggests that the two halves of the heterotetramer are relate d by a crystallographic twofold axis.