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

The wild-type phytases from the Aspergillus niger strains NRRL 3135 and T21 3 display a three-fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all o ver the sequence of the protein. Of the 12 divergent positions, three are l ocated 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 i nteract with a phosphate group of phytic acid, The fact that this presumed ionic interaction - causing stronger binding of substrates and products - c orrelates with a lower specific activity indicates that product (myo-inosit ol pentakisphosphate) release is the rate-limiting step of the reaction, (C ) 2000 Federation of European Biochemical Societies.