Structure of dihydroorotate dehydrogenase B: electron transfer between two flavin groups bridged by an iron-sulphur cluster.
BACKGROUND
The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD). Based on their sequences, DHODs are grouped into two major families. Lactococcus lactis is one of the few organisms with two DHODs, A and B, belonging to each of the two subgroups of family 1. The B enzyme (DHODB) is a prototype for DHODs in Gram-positive bacteria that use NAD+ as the second substrate. DHODB is a heterotetramer composed of two different proteins (PyrDB and PyrK) and three different cofactors: FMN, FAD, and a [2Fe-2S] cluster.


RESULTS
Crystal structures have been determined for DHODB and its product complex. The DHODB heterotetramer is composed of two closely interacting PyrDB-PyrK dimers with the [2Fe-2S] cluster in their interface centered between the FMN and FAD groups. Conformational changes are observed between the complexed and uncomplexed state of the enzyme for the loop carrying the catalytic cysteine residue and one of the lysines interacting with FMN, which is important for substrate binding.


CONCLUSIONS
A dimer of two PyrDB subunits resembling the family 1A enzymes forms the central core of DHODB. PyrK belongs to the NADPH ferredoxin reductase superfamily. The binding site for NAD+ has been deduced from the similarity to these proteins. The orotate binding in DHODB is similar to that in the family 1A enzymes. The close proximity of the three redox centers makes it possible to propose a possible electron transfer pathway involving residues conserved among the family 1B DHODs.
Rowland
P
P
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark.
Nørager
S
S
Jensen
K F
KF
Larsen
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport; D013293:Enterococcus faecalis / Q000201:enzymology; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology; D008969:Molecular Sequence Data; D009963:Orotic Acid / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000090722:Dihydroorotate Dehydrogenase; D005415:Flavins; D007506:Iron-Sulfur Proteins; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D009963:Orotic Acid; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C028878:ferredoxin-NAD+ reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors