Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase.
Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Escherichia coli carrying the pyrD gene on a multicopy plasmid. The preparation had a specific activity of 120 mumol min-1 mg-1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr = 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors. The complete sequence of 1357 base pairs of an EcoRI-EcoRI DNA fragment containing the pyrD gene was established. Dihydroorotate dehydrogenase is encoded by a 336-triplets open reading frame. The molecular mass (Mr = 36732), the amino acid composition and the N-terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292-303, i.e. Ile-Ile-Gly-Val-Gly-Gly-Ile-Asp-Ser-Val-Ile-Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed. By the S1-nuclease technique an mRNA start was mapped 34 +/- 2 nucleotide residues upstream of the beginning of the coding frame of pyrD. The leader region contains no similarity to the attenuators of the pyrB and pyrE genes of E. coli.[1]References
- Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase. Larsen, J.N., Jensen, K.F. Eur. J. Biochem. (1985) [Pubmed]
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