Citrate synthase from the thermophilic archaebacteria <i>Thermoplasma acidophilum</i> and <i>Sulfolobus acidocaldarius</i>

Smith, L.Dennis; Stevenson, Kenneth J.; Hough, David W.; Danson, Michael J.

doi:10.1016/0014-5793(87)81174-2

Cited by 14 publications

(4 citation statements)

References 22 publications

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“…acidophilum citrate synthase was expressed in E. co/i at a specific activity of up to 5 units/mg protein, that is at a level up to 20 times higher than that found in native Tp, acidophilum cells [8,9]. Production amounted to maximally lOolo of total E. coli cell protein.…”

Section: Resultsmentioning

confidence: 99%

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Expression and purification of plasmid‐encoded Thermoplasma acidophilum citrate synthase from Escherichia coli

et al. 1991

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The citrate synthase gene from the thermophilic archaebacterium. Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight. Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E. coli ribosome. A rapid purification of the expression of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

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Section: Resultsmentioning

confidence: 99%

“…The subunit molecular weight of the pure protein (43 000) corresponds to the molecular weight predicted from the amino acid sequence derived from the gene sequence (42 942) [3) and that obtained for the enzyme purified from Tp. acidophilum (43 000) [9]. Table 1 sumnaariscs the steps involved in the purification procedure.…”

Section: Resultsmentioning

confidence: 99%

Expression and purification of plasmid‐encoded Thermoplasma acidophilum citrate synthase from Escherichia coli

et al. 1991

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“…This is preceded by an ATG triplet which we propose acts as an initiation codon. The open reading frame encodes a protein of 384 amino acids (MI = 42942), corresponding to the size determined for the purified protein by SDSjPAGE [ M , = 43000 (+2000)] [20]. The amino acid composition of the citrate synthase is given in Table 1 and the codon usage for the gene is given in Table 2.…”

Section: The Sequence Of Citrate Synthasementioning

confidence: 99%

“…These organisms are thought to constitute a phylogenetically distinct evolutionary group, in addition to [3,191. The enzyme has subsequently been purified from the thermoacidophilic archaebacterium Thermoplasma acidophilum, shown to be a dimer (subunit M , = 43000) and the first 16 amino acids of the N-terminus determined [20]. In the present paper, using this amino acid sequence to design oligonucleotide probes, we report the cloning and DNA sequencing of the Tp.…”

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Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilum

Sutherland

Henneke

Towner

et al. 1990

European Journal of Biochemistry

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The gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, Thermoplasma acidophilum. We report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. The similarity is < 30% between the archaebacterial and non-archaebacterial sequences, although the majority of residues implicated in the catalytic action of the enzyme have been conserved across all three kingdoms. The cloned archaebacterial gene has been expressed in Escherichia coli to produce catalytically active citrate synthase. This is the first reported sequence of citrate synthase from the archaebacteria.Citrate synthase, the first enzyme of the citric acid cycle, has been studied from organisms that represent all three primary kingdoms : the eukaryotes, the eubacteria and the archaebacteria [l -31. Two oligomeric forms of citrate synthase have been identified: a dimeric form found in eukaryotes and Gram-positive eubacteria, and a hexameric form found in Gram-negative eubacteria. Both types are made up of identical subunits, with M , values of approximately 50 000, the hexameric form appearing to behave functionally as a trimer of the basic dimer [4]. However, the two forms of the enzyme differ in their regulatory sensitivities: the dimer is inhibited isosterically by ATP whereas the hexameric form is inhibited allosterically by NADH and, in some cases, additionally by 2-oxoglutarate [l -31.The primary amino acid sequences of citrate synthase from the eukaryotes pig heart [5] and kidney [6], Arabidopsis thaliana [7] and Saccharomyces cerevisiae [8], and from the eubacteria Escherichia coli [9, lo], Rickettsia prowazekii [l 11, Acinetobacter anitratum [12] and Pseudomonas aeruginosa [ 131 have been determined, either by amino acid sequencing of the protein or from the DNA sequence encoding the gene. In addition, a high-resolution X-ray crystallographic structure is available for the pig heart enzyme [14, 151. This has allowed identification of 12 residues involved in the active site; multiple sequence comparisons [16] have indicated that the majority of these 12 active site residues are conserved between all citrate synthases.To extend our studies on the diversity of citrate synthases and the correlation of oligomeric structure and function, we have initiated investigations on the enzyme from the archaebacteria. These organisms are thought to constitute a phylogenetically distinct evolutionary group, in addition to Correspondence to M. J. Danson, Department of Biochemistry, Enzyme. Citrate synthase (EC 4.1.3.7). Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X5.5282.

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Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two‐dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis

et al. 2006

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In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on (15)N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.

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Citrate synthase from the thermophilic archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius

Cited by 14 publications

References 22 publications

Expression and purification of plasmid‐encoded Thermoplasma acidophilum citrate synthase from Escherichia coli

Expression and purification of plasmid‐encoded Thermoplasma acidophilum citrate synthase from Escherichia coli

Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilum

Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two‐dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis

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