In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 Å resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 Å in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

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

Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC alpha and OTC beta) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC beta combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC alpha combination and the other harboring the ATC I/OTC beta combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.

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Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Tts activity is inhibited by some nucleotides that compete with ATP. In contrast with the carbamoylphosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor whose duplication gave the genes coding for carbamoyl-phosphate synthetases and carbamate kinases.Keywords : carbamoyl-phosphate synthetase; hyperthermophile; deep-sea vents ; Archae; Pyrococcus.Pyrococcus ahyssi is a hyperthermophilic archaebacterium recently isolated from a deep-sea hydrothermal vent located 2000 m deep in the North-Fiji Basin [I, 21. This microorganism has been characterized as a strict anaerobic, sulfur-metabolizing archaeum, whose optimum temperature for growth is 96°C [3]. Although R abyssi grows at atmospheric pressure, its generation time decreases at high pressure and its maximal temperature for growth is increased when cultured under hydrostatic pressure ; this microorganism is therefore classified as barotolerant to barophilic [3]. Cell growth at such a high temperature raises physiological problems, e.g. the instability of some metabolites, cellular components, and enzymes. For instance, carbamoyl phosphate, the common precursor for the biosynthesis of arginine and pyrimidine nucleotides, is a very unstable compound. At 96"C, its half-life is only a few seconds 14, 51. In all organisms, carbamoyl phosphate is synthesized by carbamoyl-phosphate synthetases (CPS), enzymes which are known for their instability. Most of the eubacteria, plants like pea [6, 71, and mung bean [XI possess a single CPS that provides carbamoyl phosphate for both the arginine and pyrimidine pathways. Eucaryotic organisms, except for plants, possess two CPS enzymes, each specific for one of these two pathways (CPS-A and CPS-P or CPS I and CPS 11, respectively). Some CPS enzymes use NH, as the donor of the amino group of carbamoyl phosphate while others use glutamine. The latter enzymes contain a subunit or domain (glutamine amidotransferase) that binds glutamine, catalyses its deamination, and transfers the NH, to the CPS catalytic site. For instance, Escherichia coli CPS is composed of two subunits, a small subunit (42 kDa), which is the glutamine amidotransferase, and a large subunit (117 kDa) which is CPS [9]. In the absence of the small subunit, the large subunit of CPS, is active and uses NH: as substrate.In a previous study, aspartate transcarbamoylase (ATC), the first enzyme of the pyrimidine pathway, which uses carbamoyl phosphate as substrate, was partially purified from the cell-free extracts of P. abyssi and its catalytic and regulatory properties were determined [lo]. In the present work, the biosynthesis of carbamoyl phosphate was investigated in this hyperthermophilic archae...

Our understanding of the icy-habitat microbiome is likely limited by a lack of reliable data on microorganisms inhabiting underground ice that has accumulated inside caves. To characterize how environmental variation impacts cave ice microbial community structure, we determined the composition of total and potentially active bacterial communities along a 13,000-year-old ice core from Scarisoara cave (Romania) through 16S rRNA gene Illumina sequencing. An average of 2,546 prokaryotic gDNA operational taxonomic units (OTUs) and 585 cDNA OTUs were identified across the perennial cave ice block and analyzed in relation to the geochemical composition of ice layers. The total microbial community and the putative active fraction displayed dissimilar taxa profiles. The ice-contained microbiome was dominated by Actinobacteria with a variable representation of Proteobacteria, while the putative active microbial community was equally shared between Proteobacteria and Firmicutes. Accordingly, a major presence of Cryobacterium, Lysinomonas, Pedobacter , and Aeromicrobium phylotypes homologous to psychrotrophic and psychrophilic bacteria from various cold environments were noted in the total community, while the prevalent putative active bacteria belonged to Clostridium, Pseudomonas, Janthinobacterium, Stenotrophomonas , and Massilia genera. Variation in the microbial cell density of ice strata with the dissolved organic carbon (DOC) content and the strong correlation of DOC and silicon concentrations revealed a major impact of depositional processes on microbial abundance throughout the ice block. Post-depositional processes appeared to occur mostly during the 4,000–7,000 years BP interval. A major bacterial composition shift was observed in 4,500–5,000-year-old ice, leading to a high representation of Beta- and Deltaproteobacteria in the potentially active community in response to the increased concentrations of DOC and major chemical elements. Estimated metabolic rates suggested the presence of a viable microbial community within the cave ice block, characterized by a maintenance metabolism in most strata and growth capacity in those ice deposits with high microbial abundance and DOC content. This first survey of microbial distribution in perennial cave ice formed since the Last Glacial period revealed a complex potentially active community, highlighting major shifts in community composition associated with geochemical changes that took place during climatic events that occurred about 5,000 years ago, with putative formation of photosynthetic biofilms.

Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 104 up to 7.5 105 cells mL−1, with 59–78% viability. Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.

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