Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.     

Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12

The nucleotide sequence of the dUTPase structural gene, dut, of Escherichia coli has been determined. The DNA sequence predicts a polypeptide chain of 150 amino acid residues (mol. wt. 16 006) corresponding in size and composition to the purified dUTPase subunit. In a tentative promoter region preceding the dut gene, the -35 and -10 regions are separated by a SacI (SstI) site. Cloning of the dut gene utilization this SacI site was previously shown to reduce dut expression dramatically. The nucleotide sequence also contains a 210-codon open reading frame 106 bp downstream of dut and co-directional with dut. Previous protein synthesis experiments using dut plasmids allocated the gene of a polypeptide of mol. wt. 23 500 to this DNA region. The open reading frame thus may correspond to a protein of unknown function co-transcribed with the dut gene.     

Regulation of Escherichia coli pyrC by the purine regulon repressor protein.

The purine regulon repressor, PurR, was identified as a component of the Escherichia coli regulatory system for pyrC, the gene that encodes dihydroorotase, an enzyme in de novo pyrimidine nucleotide synthesis. PurR binds to a pyrC control site that resembles a pur regulon operator and represses expression by twofold. Mutations that increase binding of PurR to the control site in vitro concomitantly increase in vivo regulation. There are completely independent mechanisms for regulation of pyrC by purine and pyrimidine nucleotides. Cross pathway regulation of pyrC by PurR may provide one mechanism to coordinate synthesis of purine and pyrimidine nucleotides.     

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.     

Effect of fur on pyrC gene expression

The promoter region of pyrC (dihydroorotase) gene of Escherichia coli was shown to have Fur protein binding properties by gel retardation assay. In vivo regulation of the pyrC expression was studied by measuring dihydroorotase activity and beta-galactosidase level in the fur+ and fur- genetic background. The expression of chromosomal dihydroorotase activity and beta-galactosidase activity of pyrC-lacZ fusion plasmid was repressed to about 30% and 17%, respectively in the fur+ strain compared to those in the fur- strain. Divalent ions such as Fe2+ and Zn2+ were not required for the repression. PyrC expression was also reduced to one half by 1 mM uracil. The effect of uracil was independent on the fur gene.     

The primary structure of the DeoR repressor from Escherichia coli K-12.

The nucleotide sequence of the deoR gene of E. coli, which codes for the DeoR repressor, has been determined. This gene codes for a polypeptide that is 252 amino acids residues in length. Computer-assisted analysis of the nucleotide sequence strongly suggests that the DNA binding domain of the DeoR repressor is located in the N-terminal part of the protein. After the coding region there is a dyad symmetry similar to a palindromic unit present outside many structural genes on the E. coli chromosome.