Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi

Purcarea, Cristina; Evans, David R.; Hervé, Guy

doi:10.1074/jbc.274.10.6122

Cited by 32 publications

(25 citation statements)

References 47 publications

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“…Whereas PALA was a potent inhibitor of carbamoyl aspartate formation from aspartate and exogenous carbamoyl phosphate, it was a relatively ineffective inhibitor of the coupled reaction, the formation of carbamoyl aspartate from carbamoyl phosphate generated in situ by CPSase. Similar results have been observed for the coupled reaction catalyzed by the mammalian multifunctional protein CAD (44), by the yeast bifunctional complex (CPSase-ATCase) (45), and by mixtures of P. abyssi ATCase and CPSase (46). This study provides the first indication that the inefficiency of PALA inhibition is not due to restricted access of the inhibitor to the ATCase active site in the CPSase-ATCase complex.…”

Section: Discussionsupporting

confidence: 83%

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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Section: Discussionsupporting

confidence: 83%

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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“…The question of how labile intermediates are utilized in organisms that live at elevated temperatures has been examined previously for carbamoyl phosphate (16). In Pyrococcus abyssi, a hyperthermophile with an optimal growth temperature of 96°C, carbamoyl phosphate having a half-life of 2 to 3 s at 96°C is utilized in pathways for the synthesis of arginine and pyrimidines.…”

Section: Discussionmentioning

confidence: 99%

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

Bera¹,

Chen²,

Smith

et al. 2000

J Bacteriol

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Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH 3 from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH 3 for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.The first two steps in de novo purine nucleotide synthesis involve reactions in which labile enzyme intermediates are transferred between distal, noncontiguous sites. In the first reaction, catalyzed by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (GPAT), NH 3 , generated by hydrolysis of glutamine at an N-terminal glutaminase domain, is sequestered from H 2 O by transfer through a 20-Å tunnel to a C-terminal PRPP site where synthesis of phosphoribosylamine (PRA) takes place (11). Channeling of NH 3 through a hydrophobic tunnel prevents its release from the enzyme and also avoids the likelihood of protonation of NH 3 to NH 4 ϩ , which is not a substrate (13). These steps are shown by equations 1 and 2, and the synthesis of glycinamide ribonucleotide (GAR) by the second enzyme, GAR synthetase (GARS), is given in equation 3.Glutamine ϩ H 2 O 3 glutamate ϩ NH 3(1)The GPAT half reactions are coupled, and NH 3 is channeled by transient forms of the enzyme, in which closing of a flexible loop upon PRPP binding activates the glutaminase domain and forms an NH 3 channel between the active sites (1, 2, 4, 11). PRA, the product of the second GPAT half reaction, is susceptible to hydrolysis to ribose 5-phosphate, with a half-life of 5 s under physiological conditions at 37°C (17). The kinetics for the synthesis of GAR by Escherichia coli GPAT and GARS were found to be inconsistent with free diffusion of PRA between enzymes and, as a result, a direct-transfer mechanism was proposed (17). Since evidence for a GPAT-GARS physical interaction was not obtained, direct PRA transfer was assumed to involve a transient channeling interaction between GPAT and GARS. The transient...

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“…Thus, in Pseudomonas putida and Pseudomonas aeruginosa, pyrB overlaps a pyrCЈ gene encoding a nonfunctional DHOase, which is required for the assembly of the functional dodecameric ATCase (49), and feedback inhibition and thermostability of ATCase from the extreme thermophilic bacterium Thermus ZO5 are conferred by coexpression of pyrB and the adjacent bbc (for "between b and c") and pyrC genes, respectively (58, 59). In hyperthermophilic bacteria and archaea, CPSase or carbamate kinase-like CPSase appears to interact physically with ATCase and its paralogue, ornithine carbamoyltransferase, from the arginine biosynthetic pathway, thereby allowing substrate channeling and protection of the thermolabile and potentially toxic carbamoylphosphate from the hot aqueous environment (35,57,43,44,40). Indeed, since the pathway includes several thermolabile and energy-rich substrates and precursors, some of which decompose into toxic degradation products, the very possibility of pyrimidine biosynthesis is not obvious in hot environments.…”

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confidence: 99%

“…In hyperthermophilic bacteria and archaea, CPSase or carbamate kinase-like CPSase appears to interact physically with ATCase and its paralogue, ornithine carbamoyltransferase, from the arginine biosynthetic pathway, thereby allowing substrate channeling and protection of the thermolabile and potentially toxic carbamoylphosphate from the hot aqueous environment (35,57,43,44,40). Indeed, since the pathway includes several thermolabile and energy-rich substrates and precursors, some of which decompose into toxic degradation products, the very possibility of pyrimidine biosynthesis is not obvious in hot environments.…”

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Genes of De Novo Pyrimidine Biosynthesis from the Hyperthermoacidophilic Crenarchaeote Sulfolobus acidocaldarius : Novel Organization in a Bipolar Operon

et al. 2002

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Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G؉C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3 end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).De novo synthesis of UMP is universally performed in six steps catalyzed by conserved enzymes in all three domains of life (Fig. 1). In lower eukaryotes, such as the yeast Saccharomyces cerevisiae, the carbamoylphosphate synthetase (CPSase) (EC 6.3.5.5) and aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) activities are fused in a single polypeptide chain, also containing an inactive version of dihydroorotase (DHOase) (EC 3.5.2.3). In animals the pyrimidine biosynthetic activities are highly organized; thus, the trifunctional CAD protein shows CPSase, ATCase, and DHOase activities assembled in a single polypeptide chain, and similarly, orotate phosphoribosyltransferase (OPRTase) (EC 2.4.2.10) and orotidine-5Ј-monophosphate decarboxylase (OMPdecase) (EC 4.1.1.23) are fused in a bifunctional protein. In bacteria and archaea the different reactions are performed by monofunctional enzymes, but some of them do interact, at least transiently. Thus, in Pseudomonas putida and Pseudomonas aeruginosa, pyrB overlaps a pyrCЈ gene encoding a nonfunctional DHOase, which is required for the assembly of the functional dodecameric ATCase (49), and feedback inhibition and thermo...

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Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi

Cited by 32 publications

References 47 publications

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

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

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

Genes of De Novo Pyrimidine Biosynthesis from the Hyperthermoacidophilic Crenarchaeote Sulfolobus acidocaldarius : Novel Organization in a Bipolar Operon

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