Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli

Abstract

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via d/ l-Ins(1,2,3,4,5)P 5, d/ l-Ins(2,3,4,5)P 4, d/ l-Ins(2,4,5)P 3 or d/ l-Ins(1,2,4)P 3, d/ l-Ins(1,2)P 2 or Ins(2,5)P 2 or d/ l-Ins(4,5)P 2 to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the d-Ins(1,2,3,5,6)P 5 isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F 1 and F 2 from wheat bran and the myo-inositol phosphates produced by fraction F 2. Biochim. Biophys. Acta 302, 326–328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,3,4,5)P 5, d-Ins(2,3,4,5)P 4, d-Ins(2,4,5)P 3, Ins(2,5)P 2 to finally Ins(2)P (notation 6/1/3/4/5).