Cold-active citrate synthase: mutagenesis of active-site residues.

A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.

Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus.

Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degrees C) and Pyrococcus furiosus (100 degrees C) are homo-dimeric enzymes that show a high degree of structural homology with each other, and thermostabilities commensurate with the environmental temperatures in which their host cells are found. A comparison of their atomic structures with citrate synthases from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we report the construction and analysis of site-directed mutants of the two citrate synthases to investigate the contribution of these interactions. Three sets of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T. acidophilum and P. furiosus enzymes were swapped; (b) mutants of the P. furiosus citrate synthase where the inter-subunit ionic network is disrupted; and (c) P. furiosus citrate synthase mutants in which the C-terminal arms that wrap around their partner subunits have been deleted. All three sets of mutant enzymes were expressed as recombinant proteins in Escherichia coli and were found to be catalytically active. Kinetic parameters and the dependence of catalytic activity on temperature were determined, and the stability of each enzyme was analysed by irreversible thermal inactivation experiments. The chimeric mutants indicate that the thermostability of the whole enzyme is largely determined by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain. Disruption of the inter-subunit ionic network and prevention of the C-terminal interactions both generated enzymes that were substantially less thermostable. Taken together, these data demonstrate the crucial importance of the subunit contacts to the stability of these oligomeric enzymes. Additionally, they also provide a clear distinction between thermostability and thermoactivity, showing that stability is necessary for, but does not guarantee, catalytic activity at elevated temperatures.

A microbiological survey of Montserrat Island hydrothermal biotopes.

In March 1996, a survey of hydrothermal sites on the island of Montserrat was carried out. Six sites (Galway's Soufrière. Gages Upper and Lower Soufrières, Hot Water Pond, Hot River, and Tar River Soufrière) were mapped and sampled for chemical, ATP, and microbial analyses. The hydrothermal Soufrière sites on the slopes of the active Chances Peak volcano exhibited temperatures up to almost 100 degrees C and were generally either mildly acidic at pH 5-7 or strongly acidic at pH 1.5-3, but with some hot streams and pools of low redox potential at pH 7-8. Hot Water Pond sites, comprising a series of heated pools near the western shoreline of the island. were neutral and saline, consistent with subsurface heating of entrained seawater. Biological activity shown by ATP analyses was greatest in near-neutral pH samples and generally decreased as acidity increased. A variety of heterotrophic and chemolithotrophic thermophilic organisms were isolated or observed in enrichment cultures. Most of the bacteria that were obtained in pure culture were familiar acidophiles and neutrophiles, but novel, iron-oxidizing species of Sulfobacillus were revealed. These species included the first mesophilic iron-oxidizing Sulfobacillus strains to be isolated and a strain with a higher maximum growth temperature (65 degrees C) than the previously described moderately thermophilic Sulfobacillus species.

Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Preliminary crystallographic studies of an extremely thermostable KDG aldolase from Sulfolobus solfataricus.

Crystals have been grown of 2-keto-3-deoxygluconate aldolase (KDG aldolase) from the hyperthermophilic archaeon Sulfolobus solfataricus that diffract to 2.2 A resolution. The enzyme catalyses the reversible aldol cleavage of 2-keto-3-dexoygluconate to pyruvate and glyceraldehyde, the third step of a modified non-phosphorylated Entner-Doudoroff pathway of glucose oxidation. S. solfataricus grows optimally at 353 K and the enzyme itself has a half-life of 2.5 h at 373 K. Knowledge of the crystal structure of KDG aldolase will further understanding of the basis of protein hyperthermostability and create a target for site-directed mutagenesis of active-site residues, with the aim of altering substrate specificity. Three crystal forms have been obtained: orthorhombic crystals of space group P2(1)2(1)2(1), which diffract to beyond 2.15 A, monoclinic crystals of space group C2, which diffract to 2.2 A, and cubic crystals of space group P4(2)32, which diffract to 3.4 A.

Protein purification by ultrafiltration using a beta-galactosidase fusion tag.

The use of beta-galactosidase (465 kDa) as a fusion tag for ultrafiltration-based protein purification has been investigated. The target protein studied was thermophilic glucose dehydrogenase (157 kDa, GDH) from Thermoplasma acidophilum. An expression vector was constructed comprising the lacZ gene fused to a factor Xa cleavage sequence that was attached to the 5' end of the GDH gene. This gene fusion was expressed in Escherichia coli JM109 to yield a soluble protein that exhibited activities for both enzymes. Cleavage of this fusion protein (622 kDa) by factor Xa gave two smaller proteins that showed individual beta-galactosidase and GDH activity. A two-stage diafiltration process for protein purification was used in an ultrafiltration stirred cell. In the first stage, a 500 kDa membrane was used to retain the fusion protein and transmit smaller E. coli host proteins. Approximately 80% of the GDH activity was retained in this step. Following cleavage, the second stage utilized a 300 kDa membrane to fractionate the beta-galactosidase and GDH. No beta-galactosidase was detected in the permeate solutions, and 97% of the GDH activity was recovered in the permeate.

An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates.

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricus cells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equally active with both d- and l-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH(4).

Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli.

Enzymes from extreme halophiles have potential as catalysts in biotransformations. We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase. Both enzymes were expressed in E. coli using the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity was obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl. Citrate synthase was recovered using dye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes. Expression of active protein was attempted both by growth of E. coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful.

Extremozymes.

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.

Preliminary crystallographic studies of citrate synthase from an Antarctic psychrotolerant bacterium.

Recombinant citrate synthase from a psychrotolerant bacterium, DS2-3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 A. Diffraction data collected on a synchrotron from a cryoprotected crystal extends to at least 2.0 A. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.

Structure, function and stability of enzymes from the Archaea.

The Archaea include microorganisms growing in some of the most extreme environments on earth. Consequently, their cellular components are remarkably stable entities and have considerable potential in the biotechnology industry. Here, we review the structure of archaeal enzymes in the context of their ability to function at extremes of temperature, salinity, pH and pressure.

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus.

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95 degrees C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme form the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme.

Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.

BACKGROUND: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55 degrees C and 100 degrees C having previously been determined. Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exit. RESULTS: We report here the first crystal structure of a cold-active enzyme, citrate synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 A. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. CONCLUSIONS: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the citrate synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active citrate synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.

Preliminary crystallographic studies of triosephosphate isomerase (TIM) from the hyperthermophilic Archaeon Pyrococcus woesei.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.

Site-directed mutagenesis and halophilicity of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii.

A homology-modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens. Analysis of the halophilic enzyme structure identified a potential K+-binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non-halophilic dihydrolipoamide dehydrogenases, E423 is only present in the halophilic enzyme. Four site-directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised. From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.

The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,.

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.

Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2-3R.

The gene encoding citrate synthase from a novel bacterial isolate (DS2-3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2-3R, are cold-active, with a temperature optimum of 31 degrees C. In addition the enzymes are rapidly inactivated at 45 degrees C, and show significant activity at 10 degrees C and below. Comparison of amino acid sequences indicates that DS2-3R citrate synthase is most closely related to the enzyme from gram-positive bacteria. The amino acid sequence of the DS2-3R enzyme shows several features previously recognised in other cold-active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.