The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two 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 (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia cold, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy 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, potassium hexacyanoferrate(III), and molecular oxygen as electron accepters but could not use NAD(+). The DHOdehase B purified from the E. coli strain 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 mol of native protein as tightly bound prosthetic groups. The delta K-enzyme was able to use NAD(+) as well as dichloroindophenol, potassium hexacyanoferrate(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 propose that the architecture of the delta K-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.