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...

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.

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology.

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.

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