The Differentially Conserved Residues of Carbamoyl-Phosphate Synthetase

Abstract

Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein. The larger of the two subunits (M r ϳ118,000) contains a pair of homologous domains of approximately 400 residues each that are ϳ40% identical in amino acid sequence. The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain ϳ79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other. The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled. True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other. Neither of the two possible chimeric proteins was structurally stable. These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent. This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues. Carbamoyl-phosphate synthetase (CPS)1 is a complicated protein. This enzyme catalyzes the formation of carbamoyl phosphate via a reaction mechanism that requires two molecules of ATP, bicarbonate, and either NH 4 ϩ or glutamine as a source of ammonia. The full reaction is presented in equation 1.

Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB -Based Oligonucleotide Microarray

“…The CPS large subunit harbors conserved domains that have internal sequence similarity and have been suggested to be evidence of ancestral gene duplications during evolution (29,30,47). For example, the CPS large subunit from E. coli contains two homologous domains (residues 1 to 400 and 553 to 933), with ϳ40% of the amino acid sequence being identical (29). All bacteria, including S. enterica subsp.…”

“…In addition, CPS has occasionally been used in phylogenetic analyses to examine the evolutionary relationships among Bacteria, Archaea, and Eukarya (31,32). The CPS large subunit harbors conserved domains that have internal sequence similarity and have been suggested to be evidence of ancestral gene duplications during evolution (29,30,47). For example, the CPS large subunit from E. coli contains two homologous domains (residues 1 to 400 and 553 to 933), with ϳ40% of the amino acid sequence being identical (29).…”

“…enterica. The carB gene encodes the large subunit (ϳ118 kDa) of CPS, which catalyzes the formation of carbamoyl-phosphate required for the biosynthesis of pyrimidine nucleotides and arginine or the urea cycle (28)(29)(30). In addition, CPS has occasionally been used in phylogenetic analyses to examine the evolutionary relationships among Bacteria, Archaea, and Eukarya (31,32).…”

“…We employed the carbamoylphosphate synthetase (CPS) large subunit (carB) gene as a detection biomarker after ascertaining its competence through genetic analysis. We surmised that the carB gene would be suitable because this encodes an enzyme vital to bacterial survival that contains both conserved and variable regions and has been already used for phylogenetic studies (29)(30)(31)(32)47). We designed specific capture probes based on the carB gene and constructed an oligonucleotide microarray for the effective and specific discrimination of three Salmonella serotypes, Choleraesuis, Enteritidis, and Typhimurium, which are the major serotypes in South Korea (25,26).…”

Direct demonstration of carbamoyl phosphate formation on the C‐terminal domain of carbamoyl phosphate synthetase

“…Given that clear identification of the functional roles of domains N and C is required for elucidation of the CPS mechanism, many previous studies have been aimed at physically separating the two domains and analyzing their function. The majority of studies have utilized eCPS since its structure has been solved (Raushel et al 1992; Guy and Evans 1996; Javid‐Majd et al 2000), but some have utilized eukaryotic CPSs (Guy and Evans 1996; Serre et al 1999). A variety of CPS domain constructs, with varying boundary positions, have been expressed and analyzed.…”