The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

Abstract. Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (P_i) from organic phosphorus compounds (P_org). Phytic acid (myo-inositol hexakisphosphate, IP₆) is an important form of P_org in many soils. The enzymatic hydrolysis of IP₆ by phytase yields available P_i and less phosphorylated inositol derivates as products. The hydrolysis of organic P compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ¹⁸O) of released P_i, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released P_i. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP₆, adenosine 5'-monophosphate (AMP) and glycerophosphate (GPO₄) as substrates. For a comparison to the δ¹⁸O of P_i released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP₆ as a substrate were prepared. During the hydrolysis of IP₆ by phytase, four of the six P_i were released, and one oxygen atom from water was incorporated into each P_i. This incorporation of oxygen from water into P_i was subject to an apparent inverse isotopic fractionation (ϵ ~ 6 to 10 ‰), which was similar to that imparted by acid phosphatase from potato during the hydrolysis of IP₆ (ϵ ~ 7 ‰), where less than three P_i were released. The incorporation of oxygen from water into P_i during the hydrolysis of AMP and GPO₄ by phytase yielded a normal isotopic fractionation (ϵ ~ −12 ‰), similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ϵ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking substrate dependency of the isotopic fractionation could be attributed to a difference in the δ¹⁸O values of the C–O–P bridging and non-bridging oxygen atoms in organic phosphate compounds.