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See detailStudy of the active site residues of a glycoside hydrolase family 8 xylanase
Collins, T.; De Vos, D.; Hoyoux, A. et al

in Journal of Molecular Biology (2005), 354(2), 425-435

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with ... [more ▼]

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family. (c) 2005 Elsevier Ltd. All rights reserved. [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 detailNovel xylanases and their use
Georis, Jacques; Dauvrin, Thierry; Hoyoux, Anne et al

Patent (2005)

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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 detailStructure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering
Violot, S.; Aghajari, N.; Czjzek, M. et al

in Journal of Molecular Biology (2005), 348(5), 1211-1224

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of ... [more ▼]

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of algae, into an immediate nutritive form by biodegrading this biopolymer. To understand the mechanisms of cold adaptation of its enzymatic components, we studied the structural properties of an endoglucanase, Cel5G, by complementary methods, X-ray crystallography and small angle X-ray scattering. Using X-ray crystallography, we determined the structure of the catalytic core module of this family 5 endoglucanase, at 1.4 angstrom resolution in its native form and at 1.6 angstrom in the cellobiose-bound form. The catalytic module of Cel5G presents the (beta/alpha)(8)-barrel structure typical of clan GH-A of glycoside hydrolase families. The structural comparison of the catalytic core of Cel5G with the mesophilic catalytic core of Cel5A from Erwinia chrysanthemi revealed modifications at the atomic level leading to higher flexibility and thermolability, which might account for the higher activity of Cel5G at low temperatures. Using small angle X-ray scattering we further explored the structure at the entire enzyme level. We analyzed the dimensions, shape, and conformation of Cel5G full length in solution and especially of the linker between the catalytic module and the cellulose-binding module. The results showed that the linker is unstructured, and unusually long and flexible, a peculiarity that distinguishes it from its mesophilic counterpart. Loops formed at the base by disulfide bridges presumably add constraints to stabilize the most extended conformations. These results suggest that the linker plays a major role in cold adaptation of this psychrophilic enzyme, allowing steric optimization of substrate accessibility. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

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See detailXylanases, xylanase families and extremophilic xylanases
Collins, T.; Gerday, Charles ULg; Feller, Georges ULg

in FEMS Microbiology Reviews (2005), 29(1), 3-23

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms ... [more ▼]

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. [less ▲]

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See detailActivity-stability relationships in extremophilic enzymes
D'Amico, Salvino; Collins, Tony; Georlette, Daphné et al

Poster (2005)

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See detailProtein stability
Feller, Georges ULg

Conference (2005)

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See detailEffectiveness of biostimulation techniques for cleaning of diesel polluted subAntarctic soils (Crozet Archipelago)
Delille, D.; Pelletier, E.; Coulon, F. et al

in Alleman, B. C.; Keley, M. E. (Eds.) In situ bioremediation and on site bioremediation (2005)

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See detailKinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis
Garsoux, G.; Lamotte, J.; Gerday, Charles ULg et al

in Biochemical Journal (2004), 384(Pt 2), 247-253

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and ... [more ▼]

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli. This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the Surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi. Both k(cat) and k(cat)/K-m values at 4 degreesC for the psychrophilic cellulase are similar to the values for Cel5 at 30-35 degreesC, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures. [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 detailUse of family 8 enzymes with xylanolytic activity in baking
Dutron, Agnes; Georis, Jacques; Genot, Bernard et al

Patent (2004)

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See detailAdenylation-dependent conformation and unfolding pathways of the NAD(+)-dependent DNA ligase from the thermophile Thermus scotoductus
Georlette, D.; Blaise, Vinciane ULg; Bouillenne, Fabrice ULg et al

in Biophysical Journal (2004), 86(2), 1089-1104

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these ... [more ▼]

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates. [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 detailHorizontal gene transfer from Eukarya to Bacteria and domain shuffling: the alpha-amylase model
Da Lage, J. L.; Feller, Georges ULg; Janecek, S.

in Cellular and Molecular Life Sciences : CMLS (2004), 61(1), 97-109

alpha-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal alpha ... [more ▼]

alpha-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal alpha-amylases are characterized by several typical motifs and biochemical properties. A few cases of such alpha-amylases have been previously reported in some eubacterial species. We screened the bacterial genomes available in the sequence databases for new occurrences of animal-like alpha-amylases. Three novel cases were found, which belong to unrelated bacterial phyla: Chloroflexus aurantiacus, Microbulbifer degradans, and Thermobifida fusca. All the animal-like alpha-amylases in Bacteria probably result from repeated horizontal gene transfer from animals. The M. degradans genome also contains bacterial-type and plant-type alpha-amylases in addition to the animal-type one. Thus, this species exhibits alpha-amylases of animal, plant, and bacterial origins. Moreover, the similarities in the extra C-terminal domains (different from both the alpha-amylase domain C and the starch-binding domain), when present, also suggest interkingdom as well as intragenomic shuffling. [less ▲]

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