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.

Psychrophilic, mesophilic, and thermophilic ␣-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.Our planet harbors a huge number of harsh environments that are considered as "extreme" from an anthropocentric point of view, as far as temperature, pH, osmolarity, free water, or pressure are concerned. Nevertheless, these peculiar biotopes have been successfully colonized by numerous organisms, mainly extremophilic bacteria and archaea. As the curiosity of scientists stimulates the exploration of new environments, it seems that there is no "empty space" for life on Earth and, for instance, even the supercooled cloud droplets contain actively growing bacteria (1). Among the extremophilic microorganisms, those living at extreme temperatures have attracted much attention. Thermophiles have revealed the unsuspected upper temperature for life at about 113°C (2, 3). Their enzymes have also demonstrated a considerable biotechnological potential such as the various thermostable DNA polymerases used in PCR that have boosted many laboratory techniques. At the other end of the temperature scale, metabolically active psychrophilic bacteria have been detected in liquid brine veins of sea ice at Ϫ20°C (4). These cold-loving microorganisms face the thermodynamic challenge to maintain enzyme-catalyzed reactions and metabolic rates compatible with sustained growth near or below the freezing point of pure water (5, 6). Directed evolution experiments have highlighted that, in theory, cold activity of enzymes can be gained by several subtle adjustments of the protein structure (7). However, in natural cold environments, the consensus for the adaptive strategy is to take advantage of the lack of selective pressure for stable proteins for losing stability, therefore making the enzyme more mobile or flexible at temperatures that "freeze" molecular motions and reaction rates (8).The crystal structures of extremophilic enzymes unambiguously indicate a continuum in the molecular adaptations to temperature. There is indeed a clear increase in the number and strength of all known weak interactions and structural factors involved in protein stability from psychrophiles to mesophiles (living at intermediate temperatures close to 37°C) and to thermophiles (2, 9 -1...

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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.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to EMBL under accession nos. CR954246 and CR954247.

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.

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 coldadapted 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.

The heat-labile ␣-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable ␣-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T m , the calorimetric enthalpy ⌬H cal , the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k cat and K m . The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k cat and K m by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted ␣-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation.The cold-active and heat-labile ␣-amylase from the antarctic bacterium Pseudoalteromonas haloplanktis is an exceptional case because it is the largest known protein undergoing a reversible unfolding according to a two-state reaction pathway. It has been proposed that this unusual behavior is dictated by the requirement for an improved flexibility or plasticity of the protein molecule to perform catalysis at near-zero temperatures (1). Structural investigations have suggested that this is achieved by decreasing the number and strength of weak interactions stabilizing the native conformation (2-4). Accordingly, one can expect that any mutation designed to improve the stability of this fragile edifice will induce significant perturbations of the denaturation pattern, thereby giving evidence for the structural features linked to the unfolding parameters.Small globular proteins that unfold according to a pure twostate process, i.e. reversibly and without a stable intermediate between the native and the unfolded states, have allowed researchers to establish the thermodynamic stability function accounting for the Gibbs energy change associated with their denaturation over physiological and nonphysiological temperature ranges. The current availability of this thermodynamic function, mainly gained by differential scanning calorimetry, represents much more than the simple physicochemical characterization of a polymer because it offers a powerful tool for investigation of the still unexplained "second genetic code," i.e. the driving force allowing a polypeptide to fold into a predetermined native and biologically active conformation (5). The validity of the bell-shaped stabi...

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amyloselike polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with ␣-amylases. Exactly how this enzyme catalyzes the formation of ␣-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.Amylosucrase (EC 2.4.1.4) is a glucansucrase belonging to glycoside-hydrolase (GH) 1 family 13 (1, 2). 2 This transglucosidase catalyzes the synthesis of an insoluble amylose-like polymer from sucrose (3), a cheap and easily available agroresource. This is in contrast to starch or glycogen synthases (4), which require nucleotide-activated sugar as a donor. Amylosucrase is thus attractive for the industrial synthesis of amyloselike polymers and for the modification of glucans (in particular to form nondigestible glucans) (5). Remarkably, amylosucrase is the only member of GH family 13 displaying polymerase activity and is clearly unique in this family that mainly contains starch-degrading enzymes. Amylosucrase was first isolated in the culture supernatant of Neisseria perflava (3) and later identified in various Neisseria strains (6, 7). Recently, data mining has revealed the presence of genes encoding putative amylosucrases in the genome of many other organisms such as Deinococcus radiodurans (8), Caulobacter crescentus (9), Xanthomonas campestris, Xanthomonas axonopodis (10), and Pirellula sp. (11). Recombinant amylosucrase from Neisseria polysaccharea (AS) has been the most extensively studied amylosucrase. The gene encoding AS (1) has been cloned, and its product has been purified to homogeneity. Characterization of the reaction products synthesized from sucrose substrate showed that sucrose isomers (turanose and trehalulose), glucose, maltose, and maltotriose were also produced besides the insoluble...