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M J Danson

Publications and source records attributed to M J Danson.

At least 19 recordsLinked to original sources
PubMed1994

The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum.

BACKGROUND: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 degrees C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. RESULTS: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). CONCLUSIONS: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.

Amino Acid Sequence↗
PubMed1994

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon.

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.

Amino Acid Sequence↗
PubMed1992

Zinc binding by retroviral integrase.

Zinc binding by integrase from Moloney murine leukaemia virus and a protein A fusion protein containing integrase from human immunodeficiency virus type 1 was demonstrated by a zinc blotting technique using 65ZnCl2. Autoradiography revealed a clear band that was absent from the appropriate controls. This band co-migrated with the major band in Coomassie-stained gels and in immunoblots. This binding activity was retained in the presence of competing divalent cations and was sensitive to oxidation. This is the first demonstration of zinc binding by intact retroviral integrase.

Amino Acid Sequence↗
PubMed1992

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.

Amino Acid Sequence↗
PubMed1992

The enzymology of archaebacterial pathways of central metabolism.

From a comparison of the pathways of central metabolism in the archaebacteria, eubacteria and eukaryotes, it is clear that the basic pathways were established before the divergence of the three kingdoms, but that the notable differences may provide important clues to their evolution. From these comparisons, enzymes found in all evolutionary groups have been chosen for detailed structural studies; given the range of extreme phenotypes found within the archaebacteria, these studies will be crucial to our understanding of the structural basis for protein stability and how such features may be engineered into a protein of choice.

Archaea↗
PubMed1991

Crystallization and preliminary crystallographic study of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum.

Single crystals of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group P6122 or P6522, with unit cell dimensions a = b = 121.9 angstrom, c = 229.6 angstrom and with two molecules in the asymmetric unit.

Bacterial Proteins↗
PubMed1991

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

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 product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

Base Sequence↗
PubMed1990

Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilium. Cloning and sequencing of the gene.

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 less than 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.

Amino Acid Sequence↗
PubMed1990

Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei.

In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661-665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane. The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T. brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.

Animals↗
PubMed1989

Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/- 3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E.

Amino Acid Sequence↗
PubMed1989

Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure.

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

Bacterial Proteins↗
PubMed1989

Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices.

The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids. The alignment studies have been performed as the first step in a project aimed at homology modelling E. coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes. The effects of several important variables in producing realistic alignments have been investigated. The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E. coli proteins was explored. The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated. The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.

Algorithms↗
PubMed1988

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.

Animals↗
PubMed1988

A new spectrophotometric assay for citrate synthase and its use to assess the inhibitory effects of palmitoyl thioesters.

We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.

Acyl Coenzyme A↗