title: Kinetic and structural analysis of a bacterial protein tyrosine phosphatase-like myo-inositol polyphosphatase.
abstract: PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)-like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P-loop (HCX5R) has been observed in both open (inactive) and closed (active) conformations. Site-directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo-inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3-phosphate position of Ins P6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, and D-Ins(2,4)P2. The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. Structural studies at physiological pH and high salt concentrations demonstrate the "closed" or active P-loop conformation can be induced in the absence of substrate. These results suggest PhyAsr should be reclassified as a D-3 myo-inositol hexakisphosphate phosphohydrolase and suggest the PhyAsr reaction mechanism is more similar to that of PTPs than previously suspected.
authors.affiliation: Department of Biological Sciences, University of Lethbridge, Alberta, Canada.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Puhl
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gruninger
authors.forename: Ralf
authors.initials: R
authors.lastname: Greiner
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Janzen
authors.forename: Steven C
authors.initials: SC
authors.lastname: Mosimann
authors.forename: L Brent
authors.initials: LB
authors.lastname: Selinger
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010833:Phytic Acid / Q000737:chemistry / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020357:Selenomonas / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007295:Inositol Phosphates; D007294:Inositol; D010833:Phytic Acid; D017027:Protein Tyrosine Phosphatases; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase