A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme

Makoff, Andrew; Buxton, Frank P.; Radford, Alan

doi:10.1007/bf00331004

Cited by 18 publications

(15 citation statements)

References 27 publications

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“…The shallower sedimenting peak would then be the 20,00dalton fragment, whereas the deeper sedimenting peak represents aggregates of this fragment. Indeed, the ATCase from bacteria and fungi shows a strong tendency to aggregate, and the molecule from fungi has a monomer of 21,000 daltons (14,15).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis

Davidson

Patterson

1979

Proc. Natl. Acad. Sci. U.S.A.

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A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A Pyrimidine biosynthesis has been studied extensively in eukaryotes. In several organisms, the first three enzymes in the pathway-carbamoyl-phosphate synthase [CPSase; ATP:carbamate phosphotransferase (diphosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (ATCase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (DHOase; L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3)-copurify as a single multienzyme complex (1-5). In Syrian hamster cells the native enzyme is a trimer (4).When the enzyme complex purified from Syrian hamster cells or rat hepatoma cells is subjected to sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis, a single protein band is observed at 200,000-210,000 daltons (4, 6). The homogeneity of this protein has not been established. Because the three enzyme activities from rat hepatoma cells could be partially separated by sucrose gradient centrifugation in high-salt medium, Mori and Tatibana (5) concluded that the native enzyme must be composed of separate, nonidentical subunits of similar molecular weight, whereas Coleman et al. (4) concluded from binding studies with phosphonoacetyl-laspartate, an inhibitor of ATCase, and from experiments with cells that coordinately overproduce CPSase, ATCase, and DHOase that the native enzyme is composed of identical multifunctional subunits. Thus, biochemical procedures alone have not yet resolved the question about the molecular integrity or separateness of the individual enzyme activities. Such resolution is a prerequisite to formulation of appropriate testable hypotheses regarding genetic and biochemical regulation of pyrimidine biosynthesis in mammalian cells.Recently, the isolation, from the Chinese hamster ovary (CHO) cell K1, of a mutant, Urd-A, that has decreased levels of the first three enzymes of pyrimidine biosynthesis was reported (7). Certain revertants of Urd-A (B48 and D20) have levels of CPSase and DHOase activities closer to those of CHO-K1 but still have a very low level of ATCase activity (7,8). Although CPSase, ATCase, and DHOase from CHO-K1 cosediment through a glycerol gradient, the sedimentation pattern for these enzymes from Urd-A and the revertants B48 and D20 is altered considerably. The CPSase and DHOase activities still cosediment but at a much slower rate than the same enzymes from CHO-K1 (8). In addition, the ATCase activity from the mutants is clearly separated into two peaks, one lower and the other higher in the gradient than the CPSase/DHOase peak, but both sedimenting slower than the CPSase/ATCase/ DHOase complex of CHO-Ki (8).A combined biochemical and immunological approach to study the enzymes from CHO-Ki, Urd-A, and the revertants B48 and D20 is described here. By isolating the enzymes CPSase, ATCase, and DHOase from these cell types with an antibody and p...

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Section: Discussionmentioning

confidence: 99%

“…A model for the structure of this multifunctional protein has been described for Neurospora (15), in which only the CPSase and ATCase are on one polypeptide, and for CHO cells (8). A feature common to both models is the existence of a proteasesensitive bridging sequence between the ATCase catalytic site and the rest of the protein.…”

Section: Discussionmentioning

confidence: 99%

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis

Davidson

Patterson

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…A stable, ammonia-dependent carbamoyl-phosphate synthase (CPS) was 'found with a specific activity of 2.14 f 0.53 (n = 4)nmol~min-' *mg protein-' but its inactivity with glutamine and lack of inhibition by pyrimidine nucleotides suggests that it is similar to the' arginine-specific mammalian CPS I rather than the pyrimidine-specific CPS II [24]. Aspartate carbamoyltransferase activity at a specific activity of 5.70 f 1.31 (n = 4)nmol~min-'emg protein-' was also detected in 1'.…”

Section: Febs Lettersmentioning

confidence: 99%

Pyrimidine metabolism in Trichomonas vaginalis

1984

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Pyrimidine metabolism in Trichomonas vaginalis was investigated using washed cell suspensions of the organism with radiolabelled pyrimidine ring precursors and preformed pyrimidines. The precursors [14C]orotate, [14C]bicarbonate and [14C]aspartate were not incorporated into the pyrimidine bases of trichomonal nucleic acids, indicating that the protozoan is unable to synthesise the pyrimidine ring and is dependent on the salvage of exogenous pyrimidines. [3H]uracil, [3H]uridine, [3H]cytidine, deoxy[3H]cytidine and [3H]thymidine were all efficiently salvaged, and interconversion between cytosine and uracil nucleotides was detected. Thymidylate synthase activity was not detected, suggesting that T. vaginalis is dependent upon an exogenous supply of thymidine for TMP synthesis.

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“…The ATCase from fungi such as Saccharomyces cerevisiae [6] Neurospora crassa [7,8] and Chaetomium thermophilum [9] are also multifunctional with a similar structural organization to CAD. The proteins have glutamine dependent-carbamoyl phosphate synthetase and aspartate transcarbamoylase activity, but lack DHOase activity.…”

Section: Introductionmentioning

confidence: 99%

Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex

et al. 2020

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Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chromatography showed that P. aeruginosa ATCase is monomeric which accounts for its lack of catalytic activity since the active site is a composite comprised of residues from adjacent monomers in the trimer. Circular dichroism spectroscopy indicated that the ATCase chain adopts a structure that contains secondary structure elements although neither the ATCase nor the pDHO subunits are very stable as determined by a thermal shift assay. Formation of the complex increases the melting temperature by about 30˚C. The ATCase is strongly inhibited by all nucleotide di-and triphosphates and exhibits extreme cooperativity. Previous studies suggested that the regulatory site is located in an 11-residue extension of the amino end of the catalytic chain. However, deletion of the extensions did not affect catalytic activity, nucleotide inhibition or the assembly of the dodecamer. Nucleotides destabilized the dodecamer which probably accounts for the inhibition and apparent cooperativity of the substrate saturation curves. Contrary to previous interpretations, these results suggest that P. aeruginosa ATCase is not allosterically regulated by nucleotides.

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A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme

Cited by 18 publications

References 27 publications

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis

Pyrimidine metabolism in Trichomonas vaginalis

Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex

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