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In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Crystal structures of phosphoglycerate kinase (PGK) from the psychrophile Pseudomonas sp. TACII 18 have been determined at high resolution by X-ray crystallography methods and compared with mesophilic, thermophilic and hyperthermophilic counterparts. PGK is a two-domain enzyme undergoing large domain movements to catalyze the production of ATP from 1,3-biphosphoglycerate and ADP. Whereas the conformational dynamics sustaining the catalytic mechanism of this hinge-bending enzyme now seems rather clear, the determinants which underlie high catalytic efficiency at low temperatures of this psychrophilic PGK were unknown. The comparison of the three-dimensional structures shows that multiple (global and local) specific adaptations have been brought about by this enzyme. Together, these reside in an overall increased flexibility of the cold-adapted PGK thereby allowing a better accessibility to the active site, but also a potentially more disordered transition state of the psychrophilic enzyme, due to the destabilization of some catalytic residues.

In animals, most α-amylases are chloride-dependent enzymes. A chloride ion is required for allosteric activation and is coordinated by one asparagine and two arginine side chains. Whereas the asparagine and one arginine are strictly conserved, the main chloride binding arginine is replaced by a glutamine in some rare instances, resulting in the loss of chloride binding and activation. Amyrel is a distant paralogue of α-amylase in Diptera, which was not characterized biochemically to date. Amyrel shows both substitutions depending on the species. In Drosophila melanogaster, an arginine is present in the sequence but in Drosophila virilis, a glutamine occurs at this position. We have investigated basic enzymological parameters and the dependence to chloride of Amyrel of both species, produced in yeast, and in mutants substituting arginine to glutamine or glutamine to arginine. We found that the amylolytic activity of Amyrel is about thirty times weaker than the classical Drosophila α-amylase, and that the substitution of the arginine by a glutamine in D. melanogaster suppressed the chloride-dependence but was detrimental to activity. In contrast, changing the glutamine into an arginine rendered D. virilis Amyrel chloride-dependent, and interestingly, significantly increased its catalytic efficiency. These results show that the chloride ion is not mandatory for Amyrel but stimulates the reaction rate. The possible phylogenetic origin of the arginine/glutamine substitution is also discussed.

Summary Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Trigger factor is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. The stability of this primary folding assistant was investigated using trigger factors from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli, and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by living organisms. We show that proteins adapt their stability over the whole range of biological temperatures via adjustments of the same fundamental mechanisms, involving increases in enthalpic stabilization and decreases in unfolding rates, in parallel with the environmental temperature. Enthalpic stabilization in trigger factors is characterized by large increases in the melting temperature, T(m), ranging from 33 to 96.6 °C, associated with similarly large increases in unfolding enthalpy as revealed by differential scanning calorimetry. Stopped-flow spectroscopy shows that the folding rate constants for the three investigated proteins are similar, whereas the unfolding rate constants differ by several orders of magnitude, revealing that kinetic resistance to unfolding drives adjustments of protein stability. While the unusual stability of hyperthermophilic proteins has attracted much attention, this study indicates that they are an extreme case of a more general continuum, the other extreme being represented by natively unstable proteins from psychrophiles.

The online version of this article (doi:10.1007/s00792-016-0851-9) contains supplementary material, which is available to authorized users., Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism., This work was supported by the F.R.S-FNRS, Belgium (Fonds de la Recherche Fondamentale et Collective, contract numbers 2.4535.08, 2.4523.11 and U.N009.13 to G.F.) and by the Belgian program of Interuniversity Attraction Poles (iPros P7/44) initiated by the Federal Office for Scientific, Technical and Cultural Affairs. T.C. thanks the FCT and FEDER (POFC-COMPETE) for funding through the Investigador FCT Programme (IF/01635/2014) and project EngXyl (EXPL/BBB-BIO/1772/2013- FCOMP01-0124-FEDER-041595) as well as strategic funding via UID/ BIA/04050/2013. F.R., A.G. and A.C. were FRIA research fellows., info:eu-repo/semantics/publishedVersion

The halotolerance of a cold adapted α-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01–4.5 M). AHA showed 28% increased activity in 0.5–2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) α-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10°C positive shift in the temperature optimum, a stabilization factor of >5 for thermal inactivation and a ΔT m of 8.3°C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T m indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic α-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes.

Microbial biofilms have great negative impacts on the world’s economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete anti-biofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter, and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules. The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules and to test their use in combination with conventional antibiotics.

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 homo-arginine (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 alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# 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.

Xylanases have long been used in the baking industry for improving dough stability and flexibility and for increasing bread volume and crumb structure. Only xylanases from glycoside hydrolase families 10 and 11 appear to have been tested in this application and only those from the latter family have as yet found application. Interestingly, enzymes with a putative xylanase activity are also found in glycoside hydrolase families 5, 7, 8 and 43, but apparently these have not, as yet, been tested in baking. Baking trials were used to determine the effectiveness of a psychrophilic and a mesophilic family 8 xylanolytic enzyme as well as a psychrophilic family 10 xylanase and a currently used family 11 commercial mesophilic xylanase. The potential of family 8 xylanases as technological aids in baking was clearly demonstrated as both the psychrophilic enzyme from Pseudoalteromonas haloplanktis TAH3a and the mesophilic enzyme from Bacillus halodurans C-125 had a positive effect on loaf volume. In contrast, the psychrophilic family 10 enzyme from Cryptococcus adeliae TAE85 was found to be ineffective.

The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°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°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca 2+ 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°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°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.

The cold-adapted α-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°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 β-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.

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 °C for the psychrophilic cellulase are similar to the values for Cel5 at 30–35 °C, 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.

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.

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 alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C 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.

The various organisms thriving in extreme environments on Earth, psychrophiles (cold-loving organisms) are the most abundant in terms of biomass, diversity, and distribution. The chapter presents an overview of the biotechnological uses of psychrophiles and of their biomolecules using selected examples. Site-specific mutants of psychrophiles are a useful tool to study cold adaptation and expression of cold-active enzymes at low temperature. At the industrial level, the best-known representative of polar microorganisms is certainly the yeast Candida antarctica, as its species name unambiguously refers to the sampling origin. Antarcticine-NF3 is a glycoprotein with antifreeze properties produced by the bacterium Pseudoalteromonas antarctica, which has been patented by Spanish researchers. It was found that Antarcticine is effective for scar treatment and re-epithelialization of wounds. Most studies on hydrocarbon bioremediation in polar regions have focused on the treatment of petroleum hydrocarbons, since increased petroleum exploration increases the risk of accidental oil release. Polar plants and animals have also found diverse applications and are worth citing in the context of the present survey. Recent developments based on cold-adapted organisms and their biomolecules have clearly demonstrated the huge potential of psychrophiles.

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains. a catalytic domain stabilized by Ca2+, a,galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results su,,est that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K-m and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb. (C) 2001 Academic Press.

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH 2 -terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli , and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce α-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified α-amylase shows a molecular mass of about 50 000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other α-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic α-amylase requires both Cl-and Ca2+for its amylolytic activity. Br-is also quite effecient as an allosteric effector. The comparison of the amino acid composition with those of other α-amylases from various organisms shows that the cold α-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. Key words: α-amylase, psychrophilic microorganisms, Antarctic.

The thermal stability of the cold-active α-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of ΔHcal values after consecutive calorimetric transitions, a ΔHcal/ΔHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic α-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic α-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal condition...

A considerable fraction of life develops in the sea at temperatures lower than 15°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 P. haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The P. 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, P. haloplanktis further differs from its marine counterparts with recruitment of a plasmid origin of replication for its second chromosome.

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein.

Only class C β-lactamases were found to be synthesised by ampicillin-resistant psychrophilic bacteria collected in the Antarctic. Most of them were inducible but the induction factor varied greatly. Even in non-induced conditions, where no lysis occurred, a large proportion of the activity was found in the culture supernatant, an unusual observation with class C producers.

We performed a comparative study on the enzymological features of purified recombinant α-amylase of three species belonging to the Drosophila melanogaster species subgroup: D. melanogaster, D. erecta and D. sechellia. D. erecta an d D. sechellia are specialist species, with host plant Pandanus candelabrum (Pandanaceae) and M orinda citrifolia (Rubiaceae), respectively. The temperature o ptima were around 57–60°C for the three species. The pH optima were 7.2 for D. melanogaster, 8.2 for D. erecta and 8.5 for D. sechellia. The kcat and Km were also estimated for each species with different substrates. The specialist species D. erecta and D. sechellia display a higher affinity for starch than D. melanogaster. α-Amylase activity is higher on starch than on glycogen in all species. α-A mylases of D. erecta and D. sechellia have a higher activity on maltooligosaccharides (G6 and G7) than on starch, contrary to D. melanogaster. Such differences in the e nzymological features between the species might reflect adaptation to different ecological niches and feeding habits.

Antarctic fish of the family Channichthyidae, or icefish, represent a unique model for the study of physiological and biochemical responses to chronic hypoxia since the genes coding for hemoglobin and possible myoglobin are not expressed by these teleosts. Channichthyidae have developed outstanding cardio-vascular adaptations to accommodate the lack of these hemic pigments, most of them involving the myocardium.

A heat-labile beta-lactamase has been purified from culture supernatants of Psychrobacter immobilis A5 grown at 4 degrees C and the corresponding chromosomal ampC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C beta-lactamases. The kinetic parameters of P. immobilis beta-lactamase for the hydrolysis of some beta-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.

It is frequently overlooked that the majority (>80%) of the Earth’s biosphere is cold and permanently exposed to temperatures below 5 °C (Rodrigues & Tiedje, 2008). Such low mean temperatures mainly arise from the fact that ~70% of the Earth’s surface is covered by oceans that have a constant temperature of 4°C below 1000 m depth, irrespective of the latitude. The polar regions account for another 15%, to which the glacier and alpine regions must be added, as well as the permafrost representing more than 20% of terrestrial soils. All these low temperature biotopes have been successfully colonized by cold-adapted microorganisms, termed psychrophiles (Margesin et al., 2008). These organisms do not merely endure such low and extremely inhospitable conditions but are irreversibly adapted to these environments as most psychrophiles are unable to grow at mild (or mesophilic) temperatures. Extreme psychrophiles have been traditionally sampled from Antarctic and Arctic sites, assuming that low temperatures persisting over a geological time-scale have promoted deep and efficient adaptations to freezing conditions. In addition to ice caps and sea ice, polar regions also possess unusual microbiotopes such as porous rocks in Antarctic dry valleys hosting microbial communities surviving at -60 °C (Cary et al., 2010), the liquid brine veins between sea ice crystals harboring metabolically-active microorganisms at -20 °C (Deming, 2002) or permafrost cryopegs, i.e. salty water pockets that have remained liquid at -10 °C for about 100 000 years (Gilichinsky et al., 2005). Psychrophiles and their biomolecules also possess an interesting biotechnological potential, which has already found several applications (Margesin & Feller, 2010). Cold exerts severe physicochemical constraints on living organisms including increased water viscosity, decreased molecular diffusion rates, reduced biochemical reaction rates, perturbation of weak interactions driving molecular recognition and interaction, strengthening of hydrogen bonds that, for instance, stabilize inhibitory nucleic acid structures, increased solubility of gases and stability of toxic metabolites as well as reduced fluidity of cellular membranes (D'Amico et al., 2006; Gerday & Glansdorff, 2007; Margesin et al., 2008; Rodrigues & Tiedje, 2008). Previous biochemical studies have revealed various adaptations at the molecular level such as the synthesis of cold-active enzymes by psychrophiles or the incorporation of membrane lipids promoting homeoviscosity in cold conditions. It was shown that the high level of specific activity at low temperatures of coldadapted enzymes is a key adaptation to compensate for the exponential decrease in

The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of alpha-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.

The lack of hemoglobin and of carbonic anhydrase in the blood of icefish suggest that substantial adaptations of the acid-base balance should occur in order to ensure blood pH homeostasis. The level of peptidic histidyl and of reactive -SH groups per unit of body mass in icefish plasma are 12–13 times higher than those of Notothenia rossii , a common red-blooded Antarctic species. It is proposed that the high level of imidazole ring in icefish plasma improves the non-bicarbonate buffering capacity and that the reactive sulfhydryls are involved in a redox buffer as in some other hypoxia tolerant species. After plasma fractionation on Ultrogel AcA 34, the two main icefish serum proteins have been purified by DEAE cellulose chromatography (IFI) and by HPLC on anion exchange column (IF2). IFI has been identified as a cysteine-rich para-albumin and IF2 as an histidine-rich immunoglobulin-like protein.

The growth behavior and the proteomic response after a cold shock were investigated in the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis. Remarkably, no cold-induced proteins were observed in the proteome, whereas some key proteins were repressed. This suggests noticeable differences in the cold shock response between a true psychrophile and mesophiles.

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis were compared using two-dimensional differential in-gel electrophoresis with special reference to proteins repressed by low temperatures. Remarkably, the major cold-repressed proteins, almost undetectable at 4°C, were heat shock proteins involved in folding assistance.

The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments. The Antarctic marine environment is characterized by low temperatures that very seldom exceed 0°C. Despite the permanent low temperatures, the surface water and the pack ice zones in this region harbor a surprisingly high level of microbial activity. The most abundant bacterial populations in sea ice samples from Antarctic coastal areas belong mainly to four phylogenetic groups: the Gram-positive branch, the Flexibacter-Bacteroides-Cytophaga phylum, and the alpha and gamma subdivisions of the proteobacteria (4). Up to 50% of the identified bacterial isolates could be assigned to the gammaproteobacteria (5). It is assumed that this group of heterotrophic bacteria plays a key role in carbon cycling in this extreme environment. A prominent, cold-adapted member of this group is the obligatory marine bacterium Pseudoalteromonas haloplanktis TAC125, which was isolated from an Antarctic coastal seawa

Low temperature environments are numerous on Earth and have been successfully colonized by cold-loving organisms termed psychrophiles. Cold-adapted microorganisms can be used as cell factories for the production of unstable compounds as well as for bioremediation of polluted cold soils and wastewaters. Furthermore, their biomolecules, mainly proteins and enzymes characterized by a high catalytic activity and pronounced heat-lability, have already found useful applications in various domains such as molecular biology, medical research, industrial food or feed technologies, detergents or cosmetics.

The proteomes expressed at 4 degrees C and 18 degrees C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4 degrees C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4 degrees C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33 degrees C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature-dependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

Amongst more than 1000 isolates collected in various cold environments, the strain Arthrobacter psychrolactophilus Sp 31.3 has been selected for its ability to grow and to produce exoenzymes at low temperatures, its inability to grow at 37 degrees C, its non-halophilic character and its growth versatility on various media. This non-pathogenic strain displays a strong resistance to desiccation and storage at room temperature and is suitable for the production of freeze-dried bacterial starters. When grown in a synthetic wastewater at 10 degrees C, the strain induces a complete clarification of the turbid medium and efficiently hydrolyses proteins, starch and lipids in the broth. Furthermore, this strain has a remarkable capacity to improve the biodegradability of organic compounds in wastewater as indicated by a BOD(5)/COD ratio of 0.7.

Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart.

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum (2)S(O) conformation.

International audience; 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 1.4A resolution in its native form and at 1.6A 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.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 1.4A resolution in its native form and at 1.6A 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.

International audience; Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases 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 (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (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.

A class C beta-lactamase has been purified from the culture supernatant of the antarctic psychrophile Psychrobacter immobilis A8. This psychrophilic beta-lactamase displays a low level of thermal stability and a low optimal temperature of activity. In contrast to other cold-adapted enzymes, its level of specific activity is not higher than that of mesophilic class C beta-lactamases.

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric ( M r = 18,291), 55% identical to its Escherichia coli counterpart, and displays T m and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent K m value for dihydrofolate is 50- to 80-fold higher than for E. coli , Lactobacillus casei , and T. maritima dihydrofolate reductases, whereas the apparent K m value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency ( k cat /K m ) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCaseMab), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCaseMab displays maximal activity at rather low temperatures (23 to 25°C) 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. OTCaseMab displays higher Km values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-N-phosphonoacetyl-L-ornithine (PALO). OTCaSeMab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high Km values and the lower sensitivity to PALO. The Km for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (kcat/Km) of OTCases adapted to different temperatures showed that OTCaSeMab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the Km value for ornithine observed when the temperature is brought from 20 to 5°C. OTCaSeMab 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., SCOPUS: ar.j, info:eu-repo/semantics/published

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 A 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 A. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found.

Five psychrophilic Antarctic bacteria have been selected for their capacity to secrete exoenzymes into culture medium. These strains are able to grow from 0 to about 25° C. However, production of lipase fromMoraxella, α-amylase fromAlteromonas haloplanctis, β-lactamase fromPsychrobacter immobilis and protease fromBacillus is maximal at temperatures close to that of their environment (—2 to 4° C) and is strongly inhibited at higher temperatures. This thermal effect involves alterations in the secretory pathway in the upper range of temperatures, losses due to the enzyme thermal lability and in some cases to reduction in cell development. The apparent optimal activity temperature of these enzymes is between 30 and 40° C, i.e. about 20° C lower than that of their mesophilic counterparts.

Secretion of cold-adapted α-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 α-amylase secretion in Escherichia coli . The occurrence of two different secretion pathways in different hosts is proposed.

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 A 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 A. 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.