Biochemical Characterisation of Ornithine Carbamoyltransferase from <i>Pyrococcus Furiosus</i>

Legrain, Christianne; Villeret, Vincent; Roovers, Martine; Gigot, Daniel; Dideberg, Otto; Pierard, André; Glansdorff, Nicolas

doi:10.1111/j.1432-1033.1997.01046.x

Cited by 27 publications

(32 citation statements)

References 37 publications

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“…This is ORF 615154, which encodes ornithine carbamoyltransferase (OTCase). This enzyme has been purified from P. furiosus, its gene has been cloned and expressed, and its crystal structure is available (36,70). OTCase catalyzes the transfer of a carbamoyl group from carbamoyl phosphate to ornithine, generating citrulline in the arginine biosynthetic pathway.…”

Section: Resultsmentioning

confidence: 99%

DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus : Evidence for a New Type of Sulfur-Reducing Enzyme Complex

2001

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DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S°) on the levels of gene expression in cells grown at 95°C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S°and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S°. Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S°-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S°-grown cells, and these are also thought be involved in S°r eduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S°-grown cells, a finding consistent with the fact that growth on peptides is a S°-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S°, although the reason for this is as yet unknown. Of the 20 S°-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S°-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.Hyperthermophiles are microorganisms that grow optimally at temperatures of 80°C and higher, and most are classified as archaea (65). They are a rather diverse group with respect to their metabolic capabilities, but many of them utilize peptides as a carbon source and reduce elemental sulfur (S°) to H 2 S (66). Of these, the majority are obligately proteolytic and show little if any growth unless S°is added to the growth medium. The exceptions include some species of Pyrococcus that metabolize poly-and oligosaccharides, as well as peptides (3, 4, 15). For example, Pyrococcus furiosus grows on a disaccharide (maltose) to high cell densities in the absence of S°and produces H 2 as an end product rather than H 2 S. On the other hand, P. furiosus requires S°in the growth medium when it utilizes peptides as a carbon source (1). A comparison of the activities of ...

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Section: Resultsmentioning

confidence: 99%

DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus : Evidence for a New Type of Sulfur-Reducing Enzyme Complex

2001

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“…Cold-active enzymes are frequently thermolabile, a fact which is understandable, since they have to be flexible enough to be active at a low energy cost (14). OTCase Mab is indeed considerably less resistant to thermal inactivation (Table 3) and has a weaker conformational stability (Table 4) than its E. coli or thermophilic counterparts (32,33,45). Psychrophilic enzymes counteract the inhibitory effect of low temperatures on activity by reducing the temperature dependence of the reaction rate (low activation enthalpy).…”

Section: Discussionmentioning

confidence: 99%

Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

Xu¹,

Feller

Gerday

et al. 2003

J Bacteriol

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The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase Mab ), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase Mab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase Mab displays higher K m values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue ␦-N-phosphonoacetyl-L-ornithine (PALO). OTCase Mab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K m values and the lower sensitivity to PALO. The K m for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k cat /K m ) of OTCases adapted to different temperatures showed that OTCase Mab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K m value for ornithine observed when the temperature is brought from 20 to 5°C. OTCase Mab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.Intracellular biosynthetic enzymes are usually exposed to low substrate concentrations, in contrast to extracellular enzymes. Optimizing their catalytic efficiency (k cat /K m ) in psychrophilic (cold-adapted) organisms may thus be challenging, since improving k cat at low temperatures by decreasing the activation enthalpy may have a cost in terms of affinity for the substrate(s) of the reaction (18,35). The study of cold-active enzymes is thus an important topic in terms of physiology and metabolic evolution (for recent reviews, see references 6, 21, 25, 44, and 60).No cold-active ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) had been characterized until now. However, the presence of this enzyme in microorganisms adapted to the full range of environments compatible with life makes it an excellent candidate for investigations of protein evolution and of molecular adaptations to extreme conditions. OTCase catalyzes the conversion of ornithine and carbamoyl phosphate (CP) into citrulline and inorganic phosphate in the de novo pathway for arginine biosynthesis and in the detoxifying urea cycle. Biosynthetic and urea cycle OTCases are usually homotrimers of 33-to 40-kDa subunits (7, 9), except in the hyperthermophilic archaeon Pyrococcus furiosus, where OTCase is a dodecamer (51). In Pseudomonas aeruginosa, a dodecameric catabolic OTCase catalyzes the revers...

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“…Most anabolic OTCases form trimers of about 105 kDa. The anabolic OTCase of the hyperthermophilic archaeon Pyrococcus furiosus is a dodecamer composed of four catalytic trimers (420 kDa) (31,32), making it suitable for cryo-ET analysis.…”

Section: Table II Functional Distribution Of T Acidophilum Cytosolicmentioning

confidence: 99%

Proteomics Analysis of Thermoplasma acidophilum with a Focus on Protein Complexes

Sun

Beck

Knispel

et al. 2007

Molecular & Cellular Proteomics

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Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known. Molecular & Cellular Proteomics 6: 492-502, 2007.

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Biochemical Characterisation of Ornithine Carbamoyltransferase from Pyrococcus Furiosus

Cited by 27 publications

References 37 publications

DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus : Evidence for a New Type of Sulfur-Reducing Enzyme Complex

DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus : Evidence for a New Type of Sulfur-Reducing Enzyme Complex

Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

Proteomics Analysis of Thermoplasma acidophilum with a Focus on Protein Complexes

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