Indexed on: 15 Oct '08Published on: 15 Oct '08Published in: Applied Microbiology and Biotechnology
Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P(6)), 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 degrees C. In order to elucidate its stereospecificity of Ins P(6) 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 P(6) at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly degrading Ins P(6) to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P(5), 1D-Ins(1,2,5,6)P(4), 1D-Ins(1,2,6)P(3), and 1D-Ins(1,2)P(2) (60%) and (b) 1D-Ins(1,2,3,5,6)P(5), 1D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2)(35%).