Stereo- and regiospecificity of yeast phytases–chemical synthesis and enzymatic conversion of the substrate analogues neo- and l-chiro-inositol hexakisphosphate

Phytases are enzymes that catalyze the hydrolysis of phosphate esters in myo-inositol hexakisphosphate (phytic acid). The precise routes of enzymatic dephosphorylation by phytases of the yeast strains Saccharomyces cerevisiae and Pichia rhodanensis have been investigated up to the myo-inositol trisphosphate level, including the absolute configuration of the intermediates. Stereoselective assignment of the myo-inositol pentakisphosphates (d-myo-inositol 1,2,4,5,6-pentakisphosphate and d-myo-inositol 1,2,3,4,5-pentakisphosphate) generated was accomplished by a new method based on enantiospecific enzymatic conversion and HPLC analysis. Via conduritol B or E derivatives the total syntheses of two epimers of myo-inositol hexakisphosphate, neo-inositol hexakisphosphate and l-chiro-inositol hexakisphosphate were performed to examine the specificity of the yeast phytases with these substrate analogues. A comparison of kinetic data and the degradation pathways determined gave the first hints about the molecular recognition of inositol hexakisphosphates by the enzymes. Exploitation of the high stereo- and regiospecificity observed in the dephosphorylation of neo- and l-chiro-inositol hexakisphosphate made it possible to establish enzyme-assisted steps for the synthesis of d-neo-inositol 1,2,5,6-tetrakisphosphate, l-chiro-inositol 1,2,3,5,6-pentakisphosphate and l-chiro-inositol 1,2,3,6-tetrakisphosphate.