Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex

Souciet, Jean-Luc; Nagy, M; Gouar, Martine Le; Lacroute, François; Potier, Serge

doi:10.1016/0378-1119(89)90092-9

Cited by 102 publications

(71 citation statements)

References 40 publications

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“…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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confidence: 99%

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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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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“…This multifunctional CPSase-ATCase complex from S. cerevisiae cells contains, in addition, a defective DHOase-like domain that is not catalytically active ( Fig. 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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“…The pyrC gene for DHOase is unlinked to the other genes. In lower eukaryotes, such as Saccharomyces cerevisiae, the carA/B and pyrB genes are fused and the CPSase and ATCase activities reside as superdomains on a single bifunctional CA protein encoded by the URA2 locus; DHOase is encoded by a separate URA4 gene, although a nonfunctional DHOase-like domain is located between the CPSase and ATCase domains of the CA protein (Souciet et al, 1989). In higher eukaryotes, a functional DHOase domain is positioned between the CPSase and ATCase domains of the multifunctional CAD protein.…”

Section: Structural Organization Of Genes Of the Pyrimidine Pathway Imentioning

confidence: 99%

“…An Arg-specific, ammonia-utilizing CPSase (CPSase I, N-acetylglutamate cofactor) is localized in mitochondria (except in yeasts), along with OTCase, which utilizes carbamoyl-P in the synthesis of citrulline, an intermediate in Arg biosynthesis (Davis, 1986). A pyrimidineregulated, Gln-dependent CPSase activity (CPSase 11) resides as a superdomain on a multifunctional protein that also contains ATCase (nuclear localized yeast CA protein; Nagy et al, 1989) or both ATCase and DHOase activities (cytosolic CAD protein of higher eukaryotes; Davidson et al, 1990). In these organisms, the pyrimidine pathway is regulated by inhibition of CPSase, whereas ATCase is not regulated (Wild and Wales, 1990).…”

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Molecular Cloning and Characterization of the pyrB1 and pyrB2 Genes Encoding Aspartate Transcarbamoylase in Pea (Pisum sativum L.)

Williamson

Slocum

1994

Plant Physiol.

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Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex

Cited by 102 publications

References 40 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

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

Molecular Cloning and Characterization of the pyrB1 and pyrB2 Genes Encoding Aspartate Transcarbamoylase in Pea (Pisum sativum L.)

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