Disease relevance of ECs1440

• Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies [1].
• The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E. coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex [2].
• Comparison of the deduced amino acid sequence of L leichmannii DHOase with sequences established for other organisms yielded 46.6% identity with the corresponding Bacillus subtilis enzyme [3].
• The gene encoding dihydroorotase (DHOase) of Lactobacillus leichmannii, the third enzyme of the pyrimidine biosynthetic pathway (Genbank (EMBL) accession no X78999), was cloned by phenotypic complementation of an E coli pyrC deficient mutant after transformation with Lactobacillus leichmannii genomic library DNA [3].

High impact information on ECs1440

• The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD [1].
• 1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain [1].
• Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus [1].
• However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism [4].
• Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis [4].

Chemical compound and disease context of ECs1440

• The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced [5].

Biological context of ECs1440

• CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co(2+)-substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site [4].
• Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested [4].
• Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides [6].

Associations of ECs1440 with chemical compounds

• A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis [7].
• The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6 [6].
• Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis [7].
• Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway [5].

Other interactions of ECs1440

• These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme [4].

Analytical, diagnostic and therapeutic context of ECs1440

• We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis [7].
• This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer [8].

References