Abstract
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 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 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 deltakappa-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 deltakappa-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 deltakappa-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 delta-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.
Highlights
The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene
1 The abbreviations used are: DHOdehase A and DHOdehase B, the A and B forms of dihydroorotate dehydrogenase from L. lactis; PyrDB, (DHOdehase B) and are encoded by the pyrDa and pyrDb genes, respectively (Andersen et al, 1994). Both enzymes are able to function in pyrimidine biosynthesis, since both of the genes must be inactivated by mutation in order to impose a pyrimidine requirement on L. lactis and since either of the two genes is able to correct the pyrimidine requirement of a pyrD deletion strain of Escherichia coli (Andersen et al, 1994)
The results presented in this paper document unambiguously that the polypeptides encoded by the pyrDb and pyrK genes of L. lactis form a protein complex, termed the ␦-enzyme, since they resisted separation by chromatography on several types of columns and since they migrated as a single protein species during electrophoresis in a nondenaturing gel
Summary
Materials—Restriction endonucleases, T4 DNA ligase, and Deep Vent (exo-) DNA polymerase were bought from either New England Biolabs or Boehringer Mannheim and used as recommended by the manufacturers. The fractions with most DHOdehase activity were pooled and dialyzed for 3 h against 1 liter of 5 mM sodium phosphate, pH 6, containing 10% glycerol. After washing the column with 75 ml of Buffer A, the enzyme was eluted with Buffer A containing 0.2 M NaCl. Fractions with the most DHOdehase activity were pooled and dialyzed against 1 liter of Buffer A overnight. Fractions containing most dihydroorotate dehydrogenase activity (peaking at 0.25 M sodium phosphate) were pooled, dialyzed for 2 h against 2 liters of 5 mM sodium phosphate containing 10% glycerol (pH 6), and loaded onto a 25-ml column of Matrex Red A.
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