Summary: | Permanently cold environments, like polar regions, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently at low temperatures. We have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium Pseudoalteromonas haloplanktis. Two stabilized multiple-mutants (Mut5 and Mut5CC) have been constructed. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic homolog from pig pancreas, PPA. The three enzymes AHA, Mut5 and Mut5CC have been analyzed by differential scanning calorimetry, thermal and chemical denaturation. The flexibility has been studied by acrylamide-induced fluorescence quenching. In order to investigate the kinetic origin of the gain in stability, the kinetics of unfolding and refolding in GdmCl have been monitored at 15°C. The newly introduced weak interactions stabilized the mutants, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. In addition, the two multiple-mutants exhibit an increased optimum temperature for activity. The first results of kinetic studies show a similar refolding phase but differences between the three amylases in the unfolding phase. These results unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. Identification des facteurs structuraux responsables de l’activité aux basses températures chez une -amylase produite par une bactérie psychrophile de l’Antarctique
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