Cold-Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology

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It is noteworthy to underline the psychrophilic behavior of both the wild-type and recombinants PGK which exhibit superior catalytic properties at low and moderate temperatures and a lower optimum of temperature with respect to the mesophilic PGK The k cat thermodependence of the wild-type and the native recombinant PGK is identical, whereas the k cat values recorded for the His-tagged enzyme are much lower, although still higher than those of the yeast PGK Fig.

This clearly indicates that the His-tag peptide affects both the thermostability and the catalytic properties of the His-tagged PGK. The wild-type and the native recombinant enzymes exhibit identical kinetic parameters and display a 2-fold higher catalytic efficiency when compared with that of the mesophilic PGK. In order to analyze the structural parameters responsible for the cold adaptation of the psychrophilic PGK, a three-dimensional model was built using the known three-dimensional structures of PGK from B.

As shown in Fig. As a result, the structural model displays a very similar fold consisting of two domains of almost equal size connected by a hinge region Fig.

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Analysis of the model was focused on structural factors which possibly reduce thermostability and increase flexibility, parameters which are believed to be responsible for the higher catalytic efficiency 31 , As listed in Table III , the parameters derived from the primary structure amino acid content of psychrophilic, mesophilic, thermophilic, and hyperthermophilic PGK fail to reveal significant differences which could be attributed to temperature adaptation.

The number of Gly and Pro residues, which affect the local mobility of the chain, is not altered in the cold-active PGK. Arginine residues, which have the potential to form multiple ion pairs and H-bonds, are, however, slightly less abundant. By contrast, the lower number of salt bridges and the reduced number of aromatic interactions determined on the model of the cold-adapted enzyme are consistent with its psychrophilic character.

Psychrophilic enzymes: hot topics in cold adaptation | Nature Reviews Microbiology

Furthermore, the number of these weak interactions increases significantly from the psychrophile to the hyperthermophile. Nearly all ion pairs of the psychrophilic PGK are found at the enzyme surface.

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  7. This feature is illustrated in Fig. Comparison of the electrostatic potential at the surface of PGK. Enzyme from the psychrophilic Pseudomonas sp. The psychrophilic enzyme displays a reduced number of charges at the surface. Positive charges are in blue and negatives charges in red. The N domain left is connected to the C domain right by the hinge region and delineate the active site cleft open to the top.

    Psychrophilic enzymes: hot topics in cold adaptation

    Main structural features related to temperature adaptation in psychrophilic, mesophilic, and thermophilic PGK. Heat-induced unfolding of the Antarctic Pseudomonas and yeast PGK was monitored by fluorescence spectroscopy and differential scanning calorimetry. The temperature dependence of the fluorescence signal reveals a specific denaturation pattern for the psychrophilic enzyme. Indeed, the fluorescence signal of the yeast enzyme increases with temperature, up to the so-called hyperfluorescent intermediate previously characterized 33 , according to a sharp and apparently cooperative transition Fig.

    Cold-Adapted Organisms

    By contrast, the fluorescence signal of the psychrophilic PGK decreases with temperature and denaturation occurs over a larger temperature range Fig. This indicates distinct modifications in the solvent accessibility and in the environment of the aromatic residues during the unfolding process. In addition, the experimental data slightly deviates from the monoexponential curve corresponding to a two-state unfolding model, suggesting two denaturation steps.

    Thermal unfolding of Pseudomonas left and yeast right PGK. In e-h , the thermogram for the free enzymes is given as a dashed line for comparison. The occurrence of two well separated transitions is clearly demonstrated by DSC experiments Fig. Such a biphasic profile of the heat capacity function is accounted for by either two thermodynamic domains within the protein 34 or by the coexistence of two enzyme populations, corresponding to the free form and to an enzyme-ligand complex Electrospray mass spectroscopy confirms that the mass of the psychrophilic enzyme sample is homogenous.

    One can therefore conclude that the cold-active PGK is composed of two different calorimetric units. As shown in Table IV , the psychrophilic PGK is characterized by a heat-labile domain and a heat-stable domain in respect to the T m of the yeast enzyme. However, the total amount of heat absorbed during unfolding is similar for both PGK in the free state. Thermodynamic parameters of PGK unfolding recorded by microcalorimetry. In this conformation, the thermogram of the liganted psychrophilic PGK displays, unexpectedly, no heat-stable domain and merging of both calorimetric units Fig.

    As a result, the largest difference in stability between both investigated enzymes is found in the closed conformation Table IV. In addition, we found that the sulfate ion has a remarkable effect on PGK stability. Binding of sulfate to the psychrophilic enzyme now induces the disappearance of the heat labile domain and the merging of the two calorimetric units at the level of the heat-stable domain Fig. This work has been undertaken in order to compare the psychrophilic PGK with the well characterized PGK from mesophilic and thermophilic strains and to analyze the strategy evolved by the cold-active PGK to reach psychrophily.

    A similar operon organization for the same enzymes has been reported for E. But in the case of the two latter microorganisms a tim gene is found instead of the fda gene, indicating a closer relationship of the Antarctic Pseudomonas with E. Life at low temperatures requires that psychrophilic organisms maintain sufficiently high metabolic fluxes despite thermal inhibition of enzyme reaction rates. In the case of intracellular enzymes, adaptation to cold can be achieved either by increasing k cat, or reducing K m , or both Among the limited number of cold-adapted intracellular enzymes studied so far, heat-lability is a common feature 40 but Pseudomonas PGK is the first intracellular enzyme found to display the two main characteristics of psychrophilic enzymes: a reduced thermostability and a higher catalytic efficiency.

    The higher specific activity of psychrophilic enzymes has been commonly related to their increased flexibility, also responsible for their low stability 41 , Unexpected support for this hypothesis is provided by the His-tagged psychrophilic PGK. Indeed, whereas the enzyme is stabilized by the His-tag peptide, its turnover number is drastically reduced.

    This is the first psychrophilic enzyme mutant showing the inverse relationship between stability and reaction rate. This peptide is linked to the solvent exposed N terminus which protrudes from the bottom of the N domain in Fig. The poly-His peptide possibly interacts with oppositely charged residues on the protein surface, providing the extra stability.

    This mutant also demonstrates that the His-tag can markedly alter the enzyme properties, contrary to what is commonly assumed. In addition, it is known that the extremities of a protein are preferential sites for denaturation 20 , On the other hand, the psychrophilic PGK is devoid of linkage between its N and C extremities as a result of substitutions of the involved residues Lys to Val and Glu to Ser. The presence of such salt bridges in mesophilic and thermophilic PGK can significantly increase the overall protein thermostability and their lack in the psychrophilic enzyme would therefore weaken its stability.

    Moreover, the lack of these interactions correlates well with the occurrence of two independent unfolding domains in the psychrophilic PGK as revealed by DSC. PGK achieves its closed conformation only when both substrates are bound. This catalytically competent conformation is stabilized by a newly formed ion pair, between residues Arg 62 and Asp , upon closure of the catalytic interface.

    Whereas these two residues have been found to be totally conserved in mesophilic and thermophilic PGK, the aspartic residue is substituted in the psychrophilic PGK sequence by a threonine. Thermal unfolding recorded by differential scanning calorimetry reveals an unusual denaturation pattern for the psychrophilic enzyme as it is composed of a heat-labile and a heat-stable domain.

    However, one has to recall here that the overall stability and function of a macromolecule are governed by its least stable unit Such an organization of the psychrophilic enzyme into thermodynamic units of different stability has profound implications for our current view of protein adaptation to cold.

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    From the limited number of cold-adapted enzymes studied so far, it was concluded that they are characterized by a low conformational stability, that even multidomain proteins display a reduced stability of all calorimetric units and that they have evolved toward the lowest available stability of the native state 40 , The results obtained here for the Antarctic PGK demonstrate that this evolution can affect only one particular domain of the molecule. In this respect, two possible explanations are worth mentioning. On one hand, the concept of stabilizing domains has emerged from studies of proteins such as xylanases 44 , in which the increased stability of one domain promotes the stability of the whole molecule.

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    It is therefore tempting to consider the heat-labile unit of PGK as a destabilizing domain, providing the required flexibility around the active site in order to increase the catalytic rate at low temperature. On the other hand, a high flexibility of the entire enzyme molecule should be accompanied by a broader distribution of conformational states, leading to poor ligand binding and high K m values 4.

    Therefore, maintaining a stable and rigid domain could improve substrate binding while the labile and flexible unit could favor the reaction rate. By this way, the contradictory optimization of both kinetic parameters can possibly be realized. Finally, the finding that the sulfate activator strongly influences the structural stability of both psychrophilic and mesophilic PGK can be analyzed in the context of cold adaptation. Indeed, our results suggest that at a given step in the reaction pathway, simulated by sulfate binding, the psychrophilic PGK is able to reach a conformation close to that of its mesophilic homologues, as far as stability is concerned Table IV.

    These perspectives should stimulate further kinetic and structural studies of enzymes adapted to extreme temperatures. Dumont d'Urville in Terre Adelie. We also thank Dr. Bart Devreese for assistance in mass spectroscopy and Tony Collins for carefully reading of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges.

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    Journal of Lipid Research. Previous Section Next Section. Sources The Antarctic bacteria Pseudomonas sp. Construction of the pgk Expression Vectors The pgk gene was isolated from the 7. Enzyme Assay The enzyme activity was measured in a coupled assay with glyceraldhydephosphate dehydrogenase Roche Molecular Biochemicals by monitoring the oxidation of NADH at nm using a Kontron Uvikon spectrophotometer Thermal Unfolding Heat-induced unfolding of Pseudomonas and yeast PGK Sigma was analyzed by fluorescence spectroscopy and differential scanning calorimetry.

    Cloning and Sequencing the pgk Gene A 7. Figure 1 Multiple sequence alignment of PGK. Heterologous Expression in E.