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See detailExpression, purification, crystallization and preliminary X-ray crystallographic studies of a psychrophilic cellulase from Pseudoalteromonas haloplanktis
Violot, S.; Haser, R.; Sonan, G. et al

in Acta Crystallographica Section D-Biological Crystallography (2003), 59(Part 7), 1256-1258

The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies ... [more ▼]

The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8 Angstrom has been collected. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 135.1, b = 78.4, c = 44.1 Angstrom. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found. [less ▲]

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See detailActivity, stability and flexibility in Glycosidases adapted to extreme thermal environments
Collins, T.; Meuwis, Marie-Alice ULg; Gerday, Charles ULg et al

in Journal of Molecular Biology (2003), 328(2), 419-428

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational ... [more ▼]

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation. (C) 2003 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailMetabolic enzymes from psychrophilic bacteria: Challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi
Xu, Y.; Feller, Georges ULg; Gerday, Charles ULg et al

in Journal of Bacteriology (2003), 185(7), 2161-2168

The enzyme ornithine carbamoyltransferase (OTCase) of Motitella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity ... [more ▼]

The enzyme ornithine carbamoyltransferase (OTCase) of Motitella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25degreesC) 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 delta-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. 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 5degreesC. 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. [less ▲]

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See detailMolecular adaptations to cold in psychrophilic enzymes
Feller, Georges ULg

in Cellular and Molecular Life Sciences : CMLS (2003), 60(4), 648-662

Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere. To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms ... [more ▼]

Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere. To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms synthesize enzymes characterized by a high catalytic activity at low temperatures associated, however, with low thermal stability. Thanks to recent advances provided by Xray crystallography, protein engineering and biophysical studies, we are beginning to understand the molecular adaptations responsible for these properties which appear to be relatively diverse. The emerging picture suggests that psychrophilic enzymes utilize an improved flexibility of the structures involved in the catalytic cycle, whereas other protein regions if not implicated in catalysis may or may not be subjected to genetic drift. [less ▲]

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See detailActivity-stability relationships in extremophilic enzymes
D'Amico, Salvino ULg; Marx, J. C.; Gerday, Charles ULg et al

in Journal of Biological Chemistry (2003), 278(10), 7891-7896

Psychrophilic, mesophilic, and thermophilic alpha-amylases have been studied as regards their conformational stability, heat inactivation, irreversible unfolding, activation parameters of the reaction ... [more ▼]

Psychrophilic, mesophilic, and thermophilic alpha-amylases have been studied as regards their conformational stability, heat inactivation, irreversible unfolding, activation parameters of the reaction, properties of the enzyme in complex with a transition state analog, and structural permeability. These data allowed us to propose an energy landscape for a family of extremophilic enzymes based on the folding funnel model, integrating the main differences in conformational energy, cooperativity of protein unfolding, and temperature dependence of the activity. In particular, the shape of the funnel bottom, which depicts the stability of the native state ensemble, also accounts for the thermodynamic parameters of activation that characterize these extremophilic enzymes, therefore providing a rational basis for stability-activity relationships in protein adaptation to extreme temperatures. [less ▲]

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See detailThe structure of a cold-adapted family 8 xylanase at 1.3 angstrom resolution - Structural adaptations to cold and investigation of the active site
Van Petegem, F.; Collins, T.; Meuwis, Marie-Alice ULg et al

in Journal of Biological Chemistry (2003), 278(9), 7531-7539

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermo-stability and a higher specific activity at low and moderate temperatures. The current consensus is ... [more ▼]

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermo-stability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 Angstrom resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound. [less ▲]

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See detailMicrocalorimetry as applied to psychrophilic enzymes
D'Amico, Salvino ULg; Georlette, D.; Collins, T. et al

in Ladbury, J. E. (Ed.) Biocalorimetry 2: Application of Calorimetry in the Biological Sciences (2003)

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See detailDual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase
D'Amico, Salvino ULg; Gerday, Charles ULg; Feller, Georges ULg

in Journal of Biological Chemistry (2002), 277(48), 46110-46115

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties ... [more ▼]

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T-m) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated a-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degreesC as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution. [less ▲]

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See detailSecretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: Two different pathways in different hosts
Tutino, M. L.; Parrilli, E.; Giaquinto, L. et al

in Journal of Bacteriology (2002), 184(20), 5814-5817

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic ... [more ▼]

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed. [less ▲]

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See detailA novel family 8 xylanase, functional and physicochemical characterization
Collins, T.; Meuwis, Marie-Alice ULg; Stals, I. et al

in Journal of Biological Chemistry (2002), 277(38), 35133-35139

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See detailCrystallization and preliminary X-ray analysis of a xylanase from the psychrophile Pseudoalteromonas haloplanktis
Van Petegem, F.; Collins, T.; Meuwis, Marie-Alice ULg et al

in Acta Crystallographica Section D-Biological Crystallography (2002), 58(Part 9), 1494-1496

The 46 kDa xylanase from the Antarctic microorganism Pseudoalteromonas haloplanktis is an enzyme that efficiently catalyzes reactions at low temperatures. Here, the crystallization of both the native ... [more ▼]

The 46 kDa xylanase from the Antarctic microorganism Pseudoalteromonas haloplanktis is an enzyme that efficiently catalyzes reactions at low temperatures. Here, the crystallization of both the native protein and the SeMet-substituted enzyme and data collection from both crystals using synchrotron radiation are described. The native data showed that the crystals diffract to 1.3 Angstrom resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.87, b = 90.51, c = 97.23 Angstrom. SAD data collected at the peak of the selenium absorption edge proved to be sufficient to determine the heavy-atom configuration and to obtain electron density of good quality. [less ▲]

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See detailMolecular basis of cold adaptation
D'Amico, Salvino ULg; Claverie, P.; Collins, T. et al

in Philosophical Transactions of the Royal Society of London Series B-Biological Sciences (2002), 357(1423), 917-924

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include ... [more ▼]

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes. [less ▲]

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See detailStructural basis of alpha-amylase activation by chloride
Aghajari, N.; Feller, Georges ULg; Gerday, Charles ULg et al

in Protein Science : A Publication of the Protein Society (2002), 11(6), 1435-1441

To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism ... [more ▼]

To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 Angstrom, as well as the structure of the mutants Lys300Gln (2.5 Angstrom) and Lys300Arg (2.25 Angstrom). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction. [less ▲]

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See detailA colorimetric method for the determination of lipase activity in soil
Margesin, R.; Feller, Georges ULg; Hammerle, M. et al

in Biotechnology Letters (2002), 24(1), 27-33

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30 degreesC and pH 7.25 for 10 min ... [more ▼]

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30 degreesC and pH 7.25 for 10 min. After cooling on ice and centrifugation, the released p-nitrophenol is determined at 400 nm. To allow for the adsorption of p-nitrophenol onto soil, a calibration curve is prepared in the presence of soil. [less ▲]

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See detailCold adapted endoglucanase from Antarctic bacteria
Sonan, Guillaume; Feller, Georges ULg; Gerday, Charles ULg

Poster (2002)

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See detailSimilarities and partial characterization of cold adapted alpha-amylases from Antarctic and Arctic bacteria.
Gratia, Emmanuelle; D'Amico, Salvino; Yilmaz, N. et al

Poster (2002)

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See detailA cellulase from a psychrophilic microorganism: 3D structures of its native form and its complex with cellobiose
Violot, S.; Gouet, P.; Haser, Richard et al

Poster (2002)

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See detailCloning, biochemical and structural studies of an alcohol dehydrogenase from the Antarctic bacterium Moraxella sp. TAE 123
Tsigos, I.; Georlette, Daphné; Papanikolau, Y. et al

Poster (2002)

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