β-propeller phytase hydrolyzes insoluble Ca(2+)-phytate salts and completely abrogates the ability of phytate to chelate metal ions.
Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.
Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840, Korea.
Ok-Hee
OH
Kim
Young-Ok
YO
Kim
Jae-Hoon
JH
Shim
Yun-Shin
YS
Jung
Woo-Jin
WJ
Jung
Won-Chan
WC
Choi
Heeseob
H
Lee
Sang-Jun
SJ
Lee
Kyung-Kil
KK
Kim
Joong-Huck
JH
Auh
Hyeonjin
H
Kim
Jung-Wan
JW
Kim
Tae-Kwang
TK
Oh
Byung-Chul
BC
Oh
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001682:Biological Availability; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism*; D020563:Gammaproteobacteria / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000737:chemistry; D010833:Phytic Acid / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D002413:Cations, Divalent; D002614:Chelating Agents; D007295:Inositol Phosphates; C081911:inositol 2,4,6-triphosphate; D010833:Phytic Acid; D010832:6-Phytase; D002118:Calcium