Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11.
The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because 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, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.
Oh
B C
BC
Microbial Genomic Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon,
Chang
B S
BS
Park
K H
KH
Ha
N C
NC
Kim
H K
HK
Oh
B H
BH
Oh
T K
TK
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D002384:Catalysis; D004789:Enzyme Activation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism*; D011485:Protein Binding; D013210:Staphylococcus / Q000201:enzymology*
D010833:Phytic Acid; D010744:Phosphoric Monoester Hydrolases; D010832:6-Phytase; D002118:Calcium