Psychrophilic Enzymes: Molecular Basis of Cold Adaptation

peer reviewedPsychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility

Mechanical behavior of Ti-5553 alloy. Modeling of representative cells.

This work focuses on a new beta metastable titanium alloy, Ti-5553, for aeronautical applications. The goals of this study are the characterization of the two phases (alpha and beta) of this titanium alloy and the numerical modeling of representative cells of this material, which will be used to determine the appropriate microstructure.This thesis is divided into several parts. First, the numerical tools necessary to characterize this alloy and to model representative cells using the periodic homogenization theory will be presented. Secondly, the body-centered cubic beta phase will be identied. Then, the third part will concentrate on the characterization of the hexagonal close-packed alpha phase. Finally, the last part of this thesis will focus on choosing and modeling representative cells containing the phases identfied in the previous parts.The experimental tensile tests performed at different strain rates have demonstrated the necessity of using an elastic-viscous-plastic constitutive law. Guided by macroscopic (tensile and simple shear) experiments, a microscopic plasticity-based constitutive law was chosen to characterize this alloy instead of a macroscopic Norton-Hoff's constitutive one.It will be shown that the beta phase can be fully maintained in macroscopic samples at room temperature, making the characterization of the material behavior of this phase possible from macroscopic experiments. The optimized set of parameters was validated on nanoindentation tests performed in different beta grain orientations. In addition, a sensitivity analysis of several parameters from nanoindentation tests was performed and shows the importance of accurately defining some parameters, such as the exact shape of the indenter, and the negligible influence of other parameters, such as Poisson's ratio. From this study of experimental and numerical nanoindentation tests, it also appears that the orientation of the beta grain indented hardly affects the nanoindentation results.The characterization of the alpha phase was performed using nanoindentation experimental tests available for different grain orientations. This choice was influenced by the impossibility of maintaining only an alpha phase in a macroscopic Ti-5553 sample at room temperature and by the failure to represent the phase accurately from macroscopic (alpha+beta) samples. The material characterization of this phase is complex and difficulties occur when the behavior of this phase has to be characterized for different orientations by only one set of parameters.Finally, experimental microstructures were chosen and their simplied corresponding representative cells were meshed. Numerical simulations of these representative cells were performed and the influence of several parameters will be studied, such as the effect of the appearance of the alpha phase in the beta matrix and the effect of the shape of the alpha phase on the behavior of the cell

Verification of the radiochemical purity of a labelled optical isomer

Ce travail présente une méthode pour vérifier la purete radiochimique d'un isomère marqué d'acide amink, qui permet de mesurer l'importance de la contamination par l'autre variété optique. La méthode est appliquée au cas de la L‐phénylalanine tritiée. Elle consiste à ajouter le racémique inactif, puis à préparer un dipeptide avec de l'acide L‐glutamique. Les diastéréoisoméres sont préparés chromatographiquement et la radioactivité de chacun des pics est mesurée. Ce procédé, qui ne nécessite qu'un très petit échantillon, est susceptible de détecter des contaminations extrêmement faibles

Metabolic enzymes from psychrophilic bacteria: Challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

peer reviewedThe 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

Structural basis of alpha-amylase activation by chloride

peer reviewedTo 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

Psychrophilic microorganisms: challenges for life

peer reviewedThe ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments. These challenges include: reduced enzyme activity; decreased membrane fluidity; altered transport of nutrients and waste products; decreased rates of transcription, translation and cell division; protein cold- denaturation; inappropriate protein folding; and intracellular ice formation. Cold- adapted organisms have successfully evolved features, genotypic and/ or phenotypic, to surmount the negative effects of low temperatures and to enable growth in these extreme environments. In this review, we discuss the current knowledge of these adaptations as gained from extensive biochemical and biophysical studies and also from genomics and proteomics

The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase

peer reviewedThe cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified. fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes

Revisiting differences between atopic and non-atopic asthmatics: When age is shaping airway inflammatory profile

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Purification, Characterization, and Nucleotide Sequence of the Thermolabile Alpha-Amylase from the Antarctic Psychrotroph Alteromonas Haloplanctis A23

peer reviewedThe alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures