β-Propeller Phytase Hydrolyzes Insoluble Ca²⁺-Phytate Salts and Completely Abrogates the Ability of Phytate To Chelate Metal Ions

Abstract

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.

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

“…Phytate is a molecule abundant in grain that is indigestible in non‐ruminants and like tannin has anti‐nutritional effects. The mechanism behind the interference in digestion is unknown and direct binding to proteins is difficult to detect by ITC (personal observation), however, binding of phytate to biologically important divalent metal ions can be measured by ITC …”

Combined phospho‐ and glycoproteome enrichment in nephrocalcinosis tissues of phytate‐fed rats

“…Rats fed 5% phytate, which is an indigestible chelator of dietary calcium in whole grains, developed severe nephrocalcinosis as confirmed by Alizarin red S or Von Kossa staining for calcium deposits. Severe nephrocalcinosis in rats fed phyate is mainly due to severe intestinal calcium malabsorption because of strong binding of phyate to calcium ions …”

An isothermal titration calorimetry study of phytate binding to lysozyme

“…Isothermal titration calorimetry (ITC) is a technique that provides enthalpy (DH), entropy (TDS) and Gibbs free energy values (DG) of binding by measuring the change in heat between two interacting partners in solution. It has been used successfully to characterise a diverse range of binding interactions [17], including how the b-propeller phytase hydrolyses insoluble calcium phytate salts and prevents phytate from binding to metal ions [18]. More recently, ITC has been used to determine how phytate could interact with amines in the cellular medium to gain a greater understanding of its function within cells [19].…”