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The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium

Abstract : The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate binding module (CBM) by an unusually long extended and flexible linker (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the linker was sequentially shortened by protein engineering successively deleting one and two loops of this module whereas the last disulfide bridge was also suppressed by replacing the last two cysteines by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared to those of the full-length enzyme, also to those of the cold-adapted catalytic module alone and to those of the mesophilic homologous enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by small angle X-ray scattering (SAXS). The data indicate that the sequential shortening of the linker induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long linker of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure less compact but also less stable and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.
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Submitted on : Friday, April 30, 2010 - 3:59:20 PM
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Guillaume K Sonan, Véronique Receveur-Brechot, Colette Duez, Nushin Aghajari, Mirjam Czjzek, et al.. The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium. Biochemical Journal, Portland Press, 2007, 407 (2), pp.293-302. ⟨10.1042/BJ20070640⟩. ⟨hal-00478802⟩



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