The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity. (C) 1996 Academic Press, Inc

Gluconate Dehydratase from the promiscuous Entner-Doudoroff pathway in <i>Sulfolobus solfataricus</i>

AbstractAn investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 °C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner–Doudoroff pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose

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 P6(1)22 or P6(5)22, with cell dimensions a = b = 70.8, c = 307.8 Angstrom. Diffraction data collected on a synchrotron from a cryoprotected crystal extend to at least 2.0 Angstrom. Knowledge of the structure of this enzyme Rill add to the understanding of told 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.</p

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 Angstrom 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 Angstrom, monoclinic crystals of space group C2, which diffract to 2.2 Angstrom, and cubic crystals of space group P4(2)32, which diffract to 3.4 Angstrom.</p

Stepwise adaptations of citrate synthase to survival at life's extremes: from psychrophile to hyperthermophile

The crystal structure of citrate synthase from the thermophilic Archaeon Sulfolobus solfataricus (optimum growth temperature = 85 degreesC) has been determined, extending the number of crystal structures of citrate synthase from different organisms to a total of five that span the temperature range over which life exists (from psychrophile to hyperthermophile). Detailed structural analysis has revealed possible molecular mechanisms that determine the different stabilities of the five proteins. The key to these mechanisms is the precise structural location of the additional interactions. As one ascends the temperature ladder, the subunit interface of this dimeric enzyme and loop regions are reinforced by complex electrostatic interactions, and there is a reduced exposure of hydrophobic surface. These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and interfacial regions.</p

The structural basis of substrate promiscuity in glucose dehydrogenase from the hyperthermophilic archaeon <em>Sulfolobus solfataricus</em>

The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 80 degrees C and utilizes an unusual, promiscuous, non-phosphorylative Entner-Doudoroff pathway to metabolize both glucose and galactose. The first enzyme in this pathway, glucose dehydrogenase, catalyzes the oxidation of glucose to gluconate, but has been shown to have activity with a broad range of sugar substrates, including glucose, galactose, xylose, and L-arabinose, with a requirement for the glucose stereo configuration at the C2 and C3 positions. Here we report the crystal structure of the apo form of glucose dehydrogenase to a resolution of 1.8 angstrom and a complex with its required cofactor, NADP(+), to a resolution of 2.3 angstrom. A T41A mutation was engineered to enable the trapping of substrate in the crystal. Complexes of the enzyme with D-glucose and D-xylose are presented to resolutions of 1.6 and 1.5 angstrom, respectively, that provide evidence of selectivity for the beta-anomeric, pyranose form of the substrate, and indicate that this is the productive substrate form. The nature of the promiscuity of glucose dehydrogenase is also elucidated, and a physiological role for this enzyme in xylose metabolism is suggested. Finally, the structure suggests that the mechanism of sugar oxidation by this enzyme may be similar to that described for human sorbitol dehydrogenase.</p

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 P2(1) with cell dimensions a = 79.1, b = 89.2, c = 145.4 Angstrom and beta = 92.8 degrees, and diffract to at least 2.6 Angstrom. The orthorhombic crystals belong to space group P2(1)2(1)2 with a = 89.4, b = 155.9, c = 79.5 Angstrom, and diffract to 2.9 Angstrom. 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-centring. 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 the understanding of protein thermostability.</p