Mechanisms and pathways of inositol phosphate degradation: evidence from phosphate oxygen stable isotope ratios and 31P nuclear magnetic resonance spectroscopy

Jaisi, DebPhytate and its partially dephosphorylated products exist in soils as well as in aquatic systems, but the origins, chemical forms, bioavailability and mobility of these compounds are not well understood. With increasing phytate P in agricultural soils due to manure application and consequential increase in environmental concern due to elevated P in many open waters, new methods that have higher specificity to elucidate the mechanisms and pathways of phytate degradation as well as capability for tracking sources of dissolved P are required. Application of any stable isotopes to understand phytate degradation is rare while the application of phosphate oxygen isotopes is non-existent, and there are not any previous attempts made to understand the oxygen isotopic fractionation during partial degradation of any organophosphorus compounds. This research investigated the bacterial phytate degradation kinetics in Escherichia coli and Bacillus subtilis as well as characterized the kinetics and pathways of enzymatic phytate degradation by a purified wheat phytase using a combination of novel analytical tools including NMR, HPLC, and phosphate oxygen isotope ratios (??18 O p ). The expression of phytate degradation activity by E. coli is potentially due to extracellular phytase activity, while the absence of activity in B. subtilis may be due to difference in position, synthesis, and regulation of phytase activity. Enzymatic phytate degradation results show that the phytate degradation undertakes two pathways, via D-I(1,2,3,5,6)P5 , D-I(1,2,5,6)P4 , D-I(1,2,6)P 3 , and via D-I(1,2,4,5,6)P5 , D-I(1,2,5,6)P4 , D-I(1,5,6)P 3 . Isotope results show that the cleavage of the P-O bond during phytate degradation is accompanied by the introduction of one oxygen atom solely from water to the released inorganic P. Interestingly, all phosphate moieties in phytate have the same ??18 Op values, relieving the need to fully dephosphorylate inositol phosphate before measuring its isotopic composition of released phosphate. This means that tracking the original isotopic composition of inositol phosphate (with intact six phosphate moieties) can be done from its partially dephosphorylated products and this opens up the possibility of identifying original sources of partially dephosphorylated products that are still not fully degraded after they entered in soils many decades ago. Overall, these results advance fundamental understanding of biogeochemical cycling of phytate and could be used for dual purpose of identifying fate and tracking source of inositol phosphate in the environment.University of Delaware, Department of Plant and Soil SciencesM.S