Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties

FEBS Letters

Bürgin, Haimin¹  Pasamontes, Luis¹  Höfer, Sandra¹  Kostrewa, Dirk²  Rémy, Roland¹  Kronenberger, Alexandra¹  Tomschy, Andrea¹  Vogel, Kurt¹  Tessier, Michel¹  van Loon, Adolphus P.G.M¹  Wyss, Markus¹ 

[1] Biotechnology Department, Vitamins and Fine Chemicals Division, F. Hoffmann-La Roche Ltd, Business Unit VM4, Bldg. 241/865, CH-4070 Basel, Switzerland;Pharmaceutical Research – New Technologies, F. Hoffmann-La Roche Ltd, Business Unit VM4, Bldg. 241/865, CH-4070 Basel, Switzerland

关键词: Phytase;    Acid phosphatase;    Site-directed mutagenesis;    Three-dimensional structure;    Active site;    Aspergillus niger;   

DOI  :  10.1016/S0014-5793(00)01456-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The wild-type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three-fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site-directed mutagenesis of these residues in A. niger T213 phytase showed that the R297Q mutation (R in T213, Q in NRRL 3135) fully accounts for the differences in catalytic properties observed. Molecular modelling revealed that R297 may directly interact with a phosphate group of phytic acid. The fact that this presumed ionic interaction – causing stronger binding of substrates and products – correlates with a lower specific activity indicates that product (myo-inositol pentakisphosphate) release is the rate-limiting step of the reaction.

【 授权许可】

Unknown