THE B-FORM OF DIHYDROOROTATE DEHYDROGENASE FROM LACTOCOCCUS-LACTIS CONSISTS OF 2 DIFFERENT SUBUNITS, ENCODED BY THE PYRDB AND PYRK GENES, AND CONTAINS FMN, FAD, AND [FES] REDOX CENTERS

The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DH Odehase B) is encoded by the pyrDb gene. However, recent genetic evide nce has revealed that a co-transcribed gene, pyrK is needed to achieve the proper physiological function of the enzyme. We have purified DHO dehase B from two strains of Escherichia cold, which harbored either t he pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on mul ticopy plasmids. The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassi um hexacyanoferrate(III), and molecular oxygen as electron accepters b ut could not use NAD(+). The DHOdehase B purified from the E. coli str ain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the GK-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The GK-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mo l of native protein as tightly bound prosthetic groups. The delta K-en zyme was able to use NAD(+) as well as dichloroindophenol, potassium h exacyanoferrate(III), and to some extent molecular oxygen as electron accepters for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme. Based on these results and on analysis of published sequences, we pro pose that the architecture of the delta K-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.