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Stereospecificity of myo-inositol hexakisphosphate hydrolysis by a protein tyrosine phosphatase-like inositol polyphosphatase from Megasphaera elsdenii


Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P6), are of biotechnological interest for their ability to reduce the metabolically unavailable organic phosphate content of feedstuffs and to produce lower inositol polyphosphates (IPPs) for research and pharmaceutical applications. Here, the gene coding for a new protein tyrosine phosphatase (PTP)-like IPPase was cloned from Megasphaera elsdenii (phyAme), and the biochemical properties of the recombinant protein were determined. The deduced amino acid sequence of PhyAme is similar to known PTP-like IPPases (29–44% identity), and the recombinant enzyme displayed strict specificity for IPP substrates. Optimal IPPase activity was displayed at an ionic strength of 250 mM, a pH of 5.0, and a temperature of 60°C. In order to elucidate its stereospecificity of Ins P6 dephosphorylation, a combination of high-performance ion-pair chromatography and kinetic studies was conducted. PhyAme displayed a stereospecificity that is unique among enzymes belonging to this class in that it preferentially cleaved Ins P6 at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly degrading Ins P6 to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P5, 1D-Ins(1,2,5,6)P4, 1D-Ins(1,2,6)P3, and 1D-Ins(1,2)P2 (60%) and (b) 1D-Ins(1,2,3,5,6)P5, 1D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, and d/l-Ins(1,2)P2 (35%).

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L. Brent Selinger receives funding from the Natural Sciences and Engineering Research Council (NSERC) and the Advanced Foods and Materials Network (AFMNet). Thanks to L. J. Yanke, Agriculture and Agri-Food Canada (Lethbridge, Alberta), for supplying the M. elsdenii cultures. Analysis of the isomers of the individual myo-inositol phosphate derivatives by N.-G. Carlsson, Chalmers University of Technology (Göteborg, Sweden), is also gratefully acknowledged.

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Correspondence to L. Brent Selinger.

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Puhl, A.A., Greiner, R. & Selinger, L.B. Stereospecificity of myo-inositol hexakisphosphate hydrolysis by a protein tyrosine phosphatase-like inositol polyphosphatase from Megasphaera elsdenii . Appl Microbiol Biotechnol 82, 95–103 (2009).

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  • Phytase
  • Protein tyrosine phosphatase
  • myo-Inositol
  • Stereospecificity
  • Kinetics