Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain.

Simmer, James P.; Kelly, Ruth E.; Scully, Joshua L.; Grayson, Dennis R.; Rinker, Austin G.; Bergh, Sandra T.; Evans, David R.

doi:10.1073/pnas.86.12.4382

Cited by 48 publications

(28 citation statements)

References 39 publications

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“…With eubacterial and eukaryal ATCases, the highest identities for pyrB are obtained with the corresponding enterobacterial gene from Proteus vulgaris (54.7%) (11), E. coli (54.2%) (11), Serratia marcescens (53.0%) (11), Salmonella typhimurium (52.5%) (11), and Erwinia herbicola (52.7%) (11), followed very closely by a large series of ATCase genes from eukaryotes comprising yeasts (S. cerevisiae, S. pombe) (41,47), plants (Arabidopsis thaliana, L. esculentum, Pisum sativum sp.) (48,54,79), invertebrates (Dictyostelium discoideum, Caenorhabditis elegans) (18,81), and vertebrates (Squalus acanthias, Mesocricetus auratus, C. longicaudatus) (27,42,68). The lowest identity of this archaeal pyrB gene is observed with the homologous gene from Drosophila melanogaster (38.2%) (19).…”

Section: Resultsmentioning

confidence: 98%

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Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

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The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae.Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.Studies of extremophilic organisms provide information on the molecular mechanisms of biological adaptation to extreme environments. In this regard, Pyrococcus abyssi is of particular interest, being a hyperthermophilic and barophilic/barotolerant archaeon. This euryarchaebacterium, isolated from a deepsea hydrothermal vent located 2,000 m deep in the North Fiji Basin, was characterized by Erauso et al. (15,16). It is a heterotrophic and strictly anaerobic microorganism and belongs to the sulfur-metabolizing group. At atmospheric pressure, it grows at 67 to 102°C, with an optimum at 96°C. Hydrostatic pressure slightly increases the growth rate of P. abyssi as well as its optimal and maximal growth temperatures (16).Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) is the first enzyme of the pyrimidine biosynthetic pathway. It catalyzes the carbamylation of the amino group of aspartate by carbamylphosphate with formation of carbamylaspartate and phosphate. ATCase is extensively studied as a model for structure-function relationships in cooperativity and allosteric regulation mechanisms, as well as for the evolution of these mechanisms from prokaryotes to humans. The structure and properties of the Escherichia coli enzyme have been studied in detail (for reviews, see references 3, 10, 26, 32, 40, and 70).This dodecame...

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Section: Resultsmentioning

confidence: 98%

“…ATCases from other eubacterial and eukaryotic organisms are composed either of only catalytic chains, as in Bacillus subtilis (39) and Lycopersicum esculentum (54), or of catalytic chains associated with other enzymes of the pyrimidine pathway, as in Pseudomonas putida (67), Saccharomyces cerevisiae and Schizosaccharomyces pombe (41,69), or mammals (10,17,68).…”

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Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

et al. 1997

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“…The 5-kb CAD OBR (S) resides approximately 8 kb downstream of the transcriptional start site (3,38). The nucleotide sequence of the 5Ј region of the Syrian hamster CAD gene (3,38) and a plasmid containing a 6.5-kb abbreviated cDNA insert (pCAD142 [102][103][104]) have been reported elsewhere. B, BamHI; C, ClaI; N, NotI; P, PstI; R, EcoRI; S, SacI.…”

Section: Methodsmentioning

confidence: 99%

Identification of an Origin of Bidirectional DNA Replication in the Ubiquitously Expressed Mammalian CAD Gene

Kelly

Rose

Draper

et al. 1995

Molecular and Cellular Biology

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Most DNA replication origins in eukaryotes localize to nontranscribed regions, and there are no reports of origins within constitutively expressed genes. This observation has led to the proposal that there may be an incompatibility between origin function and location within a ubiquitously expressed gene. The biochemical and functional evidence presented here demonstrates that an origin of bidirectional replication (OBR) resides within the constitutively expressed housekeeping gene CAD, which encodes the first three reactions of de novo uridine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase). The OBR was localized to a 5-kb region near the center of the Syrian hamster CAD transcriptional unit. DNA replication initiates within this region in the single-copy CAD gene in Syrian baby hamster kidney cells and in the large chromosomal amplicons that were generated after selection with N-phosphonacetyl-L-aspartate, a specific inhibitor of CAD. DNA synthesis also initiates within this OBR in autonomously replicating extrachromosomal amplicons (CAD episomes) located in an N-phosphonacetyl-L-aspartate-resistant clone (5P20) of CHOK1 cells. CAD episomes consist entirely of a multimer of Syrian hamster CAD cosmid sequences (cCAD1). These data limit the functional unit of replication initiation and timing control to the 42 kb of Syrian hamster sequences contained in cCAD1. In addition, the data indicate that the origin recognition machinery is conserved across species, since the same OBR region functions in both Syrian and Chinese hamster cells. Importantly, while cCAD1 exhibits characteristics of a complete replicon, we have not detected autonomous replication directly following transfection. Since the CAD episome was generated after excision of chromosomally integrated transfected cCAD1 sequences, we propose that prior localization within a chromosome may be necessary to ''license'' some biochemically defined OBRs to render them functional.The replicon hypothesis proposes that DNA replication initiates within specific cis-acting sequences referred to as origins (8, 62) (Fig. 1). Origins interact with trans-acting factors that enable the DNA duplex to unwind into a suitable template for DNA synthesis. A direct genetic prediction of this elegant model is that the capacity for replication is transferable; replication-incompetent DNA should replicate upon introduction of an origin. Indeed, extrachromosomal replicons that follow the cellular replication program in Escherichia coli and Saccharomyces cerevisiae can be generated by inserting a small DNA fragment into replication-defective plasmids (15, 37). Mutations within sequences that confer autonomous replication (ARS) reduce initiation efficiency or prevent it from occurring (63,80,115). The prediction that proteins involved in initiation should bind to origins has been confirmed in prokaryotes (44,47,81,90,99) and mammalian DNA viruses (24,29,88), and recent data obtained for S. cerevisiae demonstrate that ARSs are footprint...

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“…1) (42). In higher eukaryotes, including Dictyostelium discoideum (14), Drosophila melanogaster (11,15) and Mesocricetus auratus (39,41), an additional enzyme activity, namely, DHOase, is found, such that the first three enzyme activities of the pyrimidine biosynthetic pathway are found in a single trifunctional protein of a molecular mass of 220 kDa (22).…”

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

et al. 1995

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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.

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Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain.

Cited by 48 publications

References 39 publications

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli

Identification of an Origin of Bidirectional DNA Replication in the Ubiquitously Expressed Mammalian CAD Gene

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

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