Cloning, expression, and structure analysis of carbamate kinase-like carbamoyl phosphate synthetase from Pyrococcus abyssi

Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100 degrees C. Carbamoyl phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form carbamoyl phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.     

Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.     

Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.     

A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis. Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli.

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi: thermostability and 1.8A resolution crystal structure of the catalytic subunit complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate

The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits. The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells. At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable. Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C. Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability. To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli. Interactions with PALA are strictly conserved. This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit. Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini. Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.     

Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 degrees C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 degrees C and during 28 h at 15 degrees C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.     

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.     

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.     

Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima

A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.     

Expression, enzymatic characterization, and high-level production of glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.     

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi. Insights into cooperative and allosteric mechanisms

Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermophilic archaeon Pyrococcus abyssi was purified from recombinant Escherichia coli cells. The enzyme has the molecular organization of class B microbial aspartate transcarbamylases whose prototype is the E. coli enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite constraints imposed by adaptation to high temperature, the transition between T- and R-states involves significant changes in the quaternary structure, which were detected by analytical ultracentrifugation. The enzyme is allosterically regulated by ATP (activator) and by CTP and UTP (inhibitors). Nucleotide competition experiments showed that these effectors compete for the same sites. At least two regulatory properties distinguish P. abyssi ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas ATP and UTP act at millimolar concentrations, CTP inhibits at micromolar concentrations, suggesting that in P. abyssi, inhibition by CTP is the major control of enzyme activity. While V(max) increased with temperature, cooperative and allosteric effects were little or not affected, showing that molecular adaptation to high temperature allows the flexibility required to form the appropriate networks of interactions. In contrast to the same enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the carbamyl phosphate analogue phosphonacetate; this difference supports the idea that in native cells ATCase interacts with carbamyl phosphate synthetase to channel the highly thermolabile carbamyl phosphate.