Nucleotide sequence of the pyr D gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase

Abstract

Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Eschrichia coli carrying the pyr D gene on a multicopy plasmid. The preparation had a specific activity of 120 μmol min⁻¹ mg⁻¹ and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (M_r= 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 pyr D gene was established. Dihydroorotate dehydrogenase is encoded by a 336‐triplets open reading frame. The molecular mass (M_r= 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 S 1‐nuclease technique an mRNA start was mapped 34 ± 2 nucleotide residues upstream of the beginning of the coding frame of pyr D. The leader region contains no similarity to the attenuators of the pyr B and pyr E genes of E. coli.