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Volume 271, Number 46, Issue of November 15, 1996 pp. 29359-29365
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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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

(Received for publication, March 5, 1996, and in revised form, August 19, 1996)

Finn Stausholm Nielsen ^§ , Paal Skytt Andersen ^¶ and Kaj Frank Jensen

From the  Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen K, Denmark and the ^¶ Biotechnological Institute, The Technical University of Denmark, DK-2800 Lyngby, Denmark and the ^§ Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

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 coli, 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 -enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The -enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors 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 -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 -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 -enzyme was able to use NAD⁺ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified -enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the -enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.

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