Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes ph ytate (myoinositol hexakisphosphate, IP6) to less phosphorylated myo-inosit ol phosphates in the presence of Ca2+.. In this report, we discuss the uniq ue Ca2+-dependent catalytic properties of the phytase and its specific subs trate requirement. Initial rate kinetic studies of the phytase indicate tha t the enzyme activity follows a rapid equilibrium ordered mechanism in whic h binding of Ca2+ to the active site is necessary for the essential activat ion of the enzyme. Ca2+ turned out to be also required for the substrate be cause the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, wh ich is not complexed with each other. can act as competitive inhibitors. Th e Ca2+-dependent catalytic activity of the enzyme was further confirmed, an d the critical amino acid residues for the binding of Ca2+ and substrate we re identified by site-specific mutagenesis studies. Isothermal titration ca lorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the V-max and V-max/ K-m consistently supported these observations by demonstrating that the enz yme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent ac tivation of enzyme and substrate was found to be different from other histi dine acid phytases that hydrolyze metal-free phytate.