title: 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 region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR 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 cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic 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 SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR 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 LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.
authors.affiliation: Laboratoire de Biochimie et Centre d'Ingénierie des Protéines, Institut de Chimie B6, Université de Liège, B-4000 Liège Sart-Tilman, Belgium.
authors.forename: Guillaume K
authors.initials: GK
authors.lastname: Sonan
authors.forename: Véronique
authors.initials: V
authors.lastname: Receveur-Brechot
authors.forename: Colette
authors.initials: C
authors.lastname: Duez
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.forename: Mirjam
authors.initials: M
authors.lastname: Czjzek
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Charles
authors.initials: C
authors.lastname: Gerday
mesh_terms: D000064:Acclimatization; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D003080:Cold Temperature; D004795:Enzyme Stability; D009154:Mutation; D011487:Protein Conformation; D031161:Pseudoalteromonas / Q000201:enzymology*
chemical_list: D002480:Cellulase