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See detailRole of lysine versus arginine in enzyme cold-adaptation: Modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis
Siddiqui, K. S.; Poljak, A.; Guilhaus, M. et al

in Proteins-Structure Function and Bioinformatics (2006), 64(2), 486-501

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be ... [more ▼]

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homoarginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active a-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in Delta H-# and decrease in K-m. To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. [less ▲]

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See detailKinetics and energetics of ligand binding determined by microcalorimetry: Insights into active site mobility in a psychrophilic alpha-amylase
D'Amico, Salvino ULg; Sohier, Jean ULg; Feller, Georges ULg

in Journal of Molecular Biology (2006), 358(5), 1296-1304

A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the ... [more ▼]

A new microcalorimetric method for recording the kinetic parameters k(cat)/K-m and K-i of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic a-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic a-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic a-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site. (c) 2006 Elsevier Ltd. All rights reserved. [less ▲]

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See detailA nondetergent sulfobetaine prevents protein aggregation in microcalorimetric studies
Collins, T.; D'Amico, Salvino ULg; Georlette, D. et al

in Analytical Biochemistry (2006), 352(2), 299-301

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See detailPsychrophilic microorganisms: challenges for life
D'Amico, Salvino ULg; Collins, T.; Marx, J. C. et al

in EMBO Reports (2006), 7(4), 385-389

The 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 ... [more ▼]

The 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. [less ▲]

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See detailCoping with cold: The genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125
Medigue, C.; Krin, E.; Pascal, G. et al

in Genome Research (2005), 15(10), 1325-1335

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the ... [more ▼]

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome Sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased Solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by A haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The A haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, A haloplanktis further differs from its marine Counterparts with recruitment of a plasmid origin of replication for its second chromosome. [less ▲]

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See detailThe active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase
Siddiqui, K. S.; Feller, Georges ULg; D'Amico, Salvino ULg et al

in Journal of Bacteriology (2005), 187(17), 6197-6205

The 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 ... [more ▼]

The 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. [less ▲]

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See detailRole of disulfide bridges in the activity and stability of a cold-active alpha-amylase
Siddiqui, K. S.; Poljak, A.; Guilhaus, M. et al

in Journal of Bacteriology (2005), 187(17), 6206-6212

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two ... [more ▼]

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity. [less ▲]

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See detailExtreme catalysts from low-temperature environments
Hoyoux, A.; Blaise, Vinciane ULg; Collins, T. et al

in Journal of Bioscience & Bioengineering (2004), 98(5), 317-330

Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0 degrees C. To maintain metabolic rates and to prosper in cold ... [more ▼]

Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0 degrees C. To maintain metabolic rates and to prosper in cold environments, these extremophilic organisms have developed a vast array of adaptations. One main adaptive strategy developed in order to cope with the reduction of chemical reaction rates induced by low temperatures is the synthesis of cold-adapted or psychrophilic enzymes. These enzymes are characterized by a high catalytic activity at low temperatures associated with a low thermal stability. A study of protein adaptation strategies suggests that the high activity of psychrophilic enzymes could be achieved by the destabilization of the active site, allowing the catalytic center to be more flexible at low temperatures, whereas other protein regions may be destabilized or as rigid as their mesophilic counterparts. Due to these particular properties, psychrophilic enzymes offer a high potential not only for fundamental research but also for biotechnological applications. [less ▲]

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See detailA perspective on cold enzymes: Current knowledge and frequently asked questions
Marx, J. C.; Blaise, Vinciane ULg; Collins, T. et al

in Cellular and Molecular Biology (2004), 50(5), 643-655

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive ... [more ▼]

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive properties of such proteins, i.e. a high specific activity and a low thermal stability, and thus, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. This review examines the impact of low temperatures on life, the diversity of adaptation to counteract these effects and gives an overview of the features proposed to account for low thermal stability and cold-activity, following the chronological order of the catalytic cycle phases. Moreover, we present an overview of recent techniques used in the analysis of the flexibility of a protein structure which is an important concept in cold-adaptation; an overview of biotechnological potential of psychrophilic enzymes and finally, a few frequently asked questions about cold-adaptation and their possible answers. [less ▲]

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See detailSome like it cold: biocatalysis at low temperatures
Georlette, D.; Blaise, Vinciane ULg; Collins, T. et al

in FEMS Microbiology Reviews (2004), 28(1), 25-42

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less ... [more ▼]

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. [less ▲]

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See detailMolecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase
Albenne, C.; Skov, L. K.; Mirza, O. et al

in Journal of Biological Chemistry (2004), 279(1), 726-734

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycosidehydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any ... [more ▼]

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycosidehydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance ( Arg(415), subsite +4) and the correct positioning (Asp(394) and Arg(446), subsite +1) of acceptor molecules. On the other hand, Arg(226) (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products. [less ▲]

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See detailActivity-Stability Relationships in Extremophilic Enzymes
D'Amico, Salvino ULg

Conference (2004)

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See detailActivity-Stability Relationships in Extremophilic Enzymes
D'Amico, Salvino ULg

Conference (2004)

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See detailTemperature adaptation of proteins: Engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase
D'Amico, Salvino ULg; Gerday, Charles ULg; Feller, Georges ULg

in Journal of Molecular Biology (2003), 332(5), 981-988

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic (alpha-amylase) with or without an extra ... [more ▼]

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic (alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature. (C) 2003 Elsevier Ltd. All rights reserved. [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 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 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 detailMicrocalorimetry as applied to psychrophilic enzymes
D'Amico, Salvino ULg

Conference (2002)

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