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

Schurr, Michael J.; Vickrey, John F.; Kumar, Amar; Campbell, Alan L.; Cunin, Raymond; Benjamin, Robert C.; Shanley, Mark S.; O’Donovan, Gerard A.

doi:10.1128/jb.177.7.1751-1759.1995

Cited by 41 publications

(45 citation statements)

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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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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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“…In contrast, previous attempts (25) to express Pseudomonas catalytic subunits by deletion of the pDHO gene (pyrCЈ) yielded plasmids that, unlike the parental constructs, were unable to complement E. coli strains deficient in ATCase. This observation led to the suggestion that, unlike the catalytic subunit of E. coli ATCase and the class C enzymes, the P. aeruginosa catalytic subunit is inactive in the absence of the pDHO chains.…”

Section: E Coli Atcase Despite the Very High Hill Coefficients (mentioning

confidence: 71%

“…In support of this interpretation, chemical modification with the ATP analog, FSBA, showed (23) that the nucleotides bind to the catalytic subunit. Moreover, deletion mutants lacking the first 34 residues at the amino end of the P. putida catalytic chain have been found (25) to be insensitive to nucleotides, indicating that this region of the molecule participates in feedback inhibition. Much of this chain segment may well be important, although 8 of these residues have been substituted in the construct described here, with the complete retention of nucleotide sensitivity.…”

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“…When O'Donovan and associates (25,45) cloned and sequenced the genes encoding the enzyme from Pseudomonas putida and P. aeruginosa, the surprising discovery was that, although the 36-kDa chains were clearly homologous to the catalytic chains of other well characterized ATCases, the sequence of the 45-kDa polypeptide closely resembled dihydroorotase, the enzyme that catalyzes the subsequent step in the de novo pyrimidine biosynthetic pathway. However, the enzyme complex lacks dihydroorotase activity, so the 45-kDa polypeptide has been designated a pseudo-DHOase, analogous to the homologous inactive domain found (26) in the yeast multifunctional protein encoded by the ura2 locus.…”

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Pseudomonas aeruginosa Aspartate Transcarbamoylase

Vickrey¹,

Hervé²,

Evans³

2002

Journal of Biological Chemistry

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Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.Pseudomonadaceae is a family of eubacteria with a wide ecological distribution that is pathogenic in animals (1, 2). Pseudomonas aeruginosa, the principle cause of morbidity and mortality in immunocompromised patients and burn victims (3-5), is an obligate pathogen. In cystic fibrosis, P. aeruginosa infection is the usual cause of death. In the design of therapeutic strategies, comparatively little attention has focused on the physiology of the organism itself, despite the unusual metabolism exhibited by Pseudomonadaceae such as uracil catabolism (6), the lack of gene repression (7,8), and the ability to use arginine as the sole source of carbon, nitrogen, and energy (9, 10).In other organisms, studies of de novo pyrimidine biosynthesis and salvage are actively pursued because of the relationship of these pathways to growth, development, and chemotherapy. Aspartate transcarbamoylase (ATCase, 1 EC 2.1.3.2) catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate (11) in the de novo biosynthetic pathway. The structure and function of the enzyme from Escherichia coli has been extensively studied and has become the prototype of allosteric enzymes. However, ATCases are highly polymorphic differing both in structure and mode of regulation. Jones and co-workers (12) identified three distinct classes of bacterial aspartate transcarbamoylase.E. coli ATCase, designated a class B enzyme, consists of six copies of two types of polypeptide chains, catalytic and regulatory. The 34-kDa catalytic chains associate to form catalytically active but unr...

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“…Bacterial ATCases have been classified into 1) Class A, 480-kDa duodecamers consisting of two catalytic trimers and six subunits that are either dihydroorotase (Thermus aquaticus (6)), the third enzyme in the pathway, or inactive dihydroorotase homologues (e.g. Pseudomonas aeruginosa ATCase (7,8)); 2) Class B, 310-kDa duodecamers consisting of two catalytic trimers and three regulatory dimers that bind allosteric effectors (e.g. Escherichia coli ATCase (9)); and 3) Class C, unregulated 100-kDa catalytic trimers (e.g.…”

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Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

Purcarea

Ahuja

et al. 2003

Journal of Biological Chemistry

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Aquifex aeolicus, an organism that flourishes at 95°C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in k cat with little change in the K m for the substrates, carbamoyl phosphate and aspartate. The K m for both substrates was 30 -40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.Aquifex aeolicus, one of the most hyperthermophilic eubacteria thus far discovered, is classified as a hydrogen-oxidizing, microaerophilic, obligate chemolithoautotroph (1). This marine organism is related to the filamentous bacteria isolated from the hot springs in Yellowstone near the turn of the last century (2, 3). One intriguing question is how unstable metabolites are preserved from thermal degradation in A. aeolicus and other hyperthermophiles. For example, carbamoyl phosphate, a key intermediate in both pyrimidine and arginine biosynthetic pathways, has a half-life of less than 2 s at 100°C and decomposes to toxic cyanate, a promiscuous alkylating agent (4, 5).In the pyrimidine biosynthetic pathway, carbamoyl phosphate is used as a substrate, along with aspartate, for the formation of carbamoyl aspartate in a reaction catalyzed by aspartate transcarbamoylase (ATCase 1 ; EC 2.1.3.2).Carbamoyl phosphate ϩ aspartate 3 carbamoyl aspartate ϩ P i REACTION 1

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

Cited by 41 publications

References 44 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

Pseudomonas aeruginosa Aspartate Transcarbamoylase

Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

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