title: Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties.
abstract: 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.
authors.affiliation: Biotechnology Department, Vitamins and Fine Chemicals Division, F. Hoffmann-La Roche Ltd, Business Unit VM4, Bldg. 241/865, CH-4070, Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Tomschy
authors.forename: M
authors.initials: M
authors.lastname: Wyss
authors.forename: D
authors.initials: D
authors.lastname: Kostrewa
authors.forename: K
authors.initials: K
authors.lastname: Vogel
authors.forename: M
authors.initials: M
authors.lastname: Tessier
authors.forename: S
authors.initials: S
authors.lastname: Höfer
authors.forename: H
authors.initials: H
authors.lastname: Bürgin
authors.forename: A
authors.initials: A
authors.lastname: Kronenberger
authors.forename: R
authors.initials: R
authors.lastname: Rémy
authors.forename: A P
authors.initials: AP
authors.lastname: van Loon
authors.forename: L
authors.initials: L
authors.lastname: Pasamontes
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D010832:6-Phytase