Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a psychrophilic alpha-Amylase

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.