title: Crucial role of Pro 257 in the thermostability of Bacillus phytases: biochemical and structural investigation.
abstract: We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VTTE-68013 by the R257P substitution. PHY US417 was shown to be more thermostable than PhyC. To elucidate the mechanism of how the Pro 257 changes the thermostability of Bacillus phytases, this residue was mutated to Arg and Ala. The experimental results revealed that the thermostability of the P257A mutants and especially P257R was significantly decreased. The P257R and P257A mutants recovered, respectively, 64.4 and 81.5% of the wild-type activity after incubation at 75 °C for 30 min in the presence of 5mM CaCl(2). The P257R mutation also led to a severe reduction in the specific activity and catalytic efficiency of the enzyme. Structural investigation, by molecular modeling of PHY US417 and PhyC focused on the region of the 257 residue, revealed that this residue was present in a surface loop connecting two of the six characteristic β sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.
authors.affiliation: Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP 1177 3018 Sfax, Tunisia.
authors.forename: Ameny
authors.initials: A
authors.lastname: Farhat-Khemakhem
authors.forename: Mamdouh Ben
authors.initials: MB
authors.lastname: Ali
authors.forename: Ines
authors.initials: I
authors.lastname: Boukhris
authors.forename: Bassem
authors.initials: B
authors.lastname: Khemakhem
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Maguin
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
authors.forename: Hichem
authors.initials: H
authors.lastname: Chouayekh
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011392:Proline; D010832:6-Phytase; D002118:Calcium