A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate.
Although it is becoming well known that myo-inositol polyphosphates and the enzymes involved in their metabolism play a critical role in eukaryotic systems, little is understood of their significance in prokaryotic systems. A novel protein tyrosine phosphatase (PTP)-like inositol polyphosphatase (IPPase) gene has been cloned from Selenomonas ruminantium subsp. lactilytica (phyAsrl). The deduced amino acid sequence of PhyAsrl is most similar to a PTP-like IPPase from the anaerobic bacterium S. ruminantium (35% identity), but also shows similarity (19-30% identity) to various other putative prokaryotic PTPs. Recombinant PhyAsrl could dephosphorylate myo-inositol hexakisphosphate (Ins P(6)) in vitro, and maximal activity was displayed at an ionic strength of 200 mM, a pH of 4.5, and a temperature of 55 degrees C. In order to elucidate its substrate specificity and pathway of Ins P(6) dephosphorylation, a combination of kinetic and high-performance ion-pair chromatography studies were conducted. The data indicated that PhyAsrl has a general specificity for polyphosphorylated myo-inositol substrates, but can also dephosphorylate molecules containing high energy pyrophosphate bonds in vitro. PhyAsrl is unique from other microbial IPPases in that it preferentially cleaves the 5-phosphate position of Ins P(6). Furthermore, it can produce Ins(2)P via a highly unique and ordered pathway of sequential dephosphorylation: Ins P(6), Ins(1,2,3,4,6)P(5), D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2). Finally, reverse transcription PCR was used to determine that phyAsrl is constitutively expressed, and together with bioinformatic analysis, was used to gain an understanding of its physiological significance.
Puhl
Aaron A
AA
Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4.
Greiner
Ralf
R
Selinger
L Brent
LB
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D010833:Phytic Acid / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020357:Selenomonas / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D010710:Phosphates; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase; D017027:Protein Tyrosine Phosphatases