Crucial role of Pro 257 in the thermostability of Bacillus phytases: Biochemical and structural investigation - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue International Journal of Biological Macromolecules Année : 2013

Crucial role of Pro 257 in the thermostability of Bacillus phytases: Biochemical and structural investigation

Résumé

We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VITE-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 degrees C for 30 min in the presence of 5 mM CaCl2. 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 13 sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.

Dates et versions

hal-01003322 , version 1 (10-06-2014)

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Ameny Farhat-Khemakhem, Mamdouh Ben Ali, Ines Boukhris, Bassem Khemakhem, Emmanuelle Maguin, et al.. Crucial role of Pro 257 in the thermostability of Bacillus phytases: Biochemical and structural investigation. International Journal of Biological Macromolecules, 2013, 54, pp.9-15. ⟨10.1016/j.ijbiomac.2012.11.020⟩. ⟨hal-01003322⟩
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