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Published online before print June 13, 2007, 10.1110/ps.062738307
Protein Science (2007), 16:1368-1378. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Kinetic and structural analysis of a bacterial protein tyrosine phosphatase-like myo-inositol polyphosphatase

Aaron A. Puhl1,4, Robert J. Gruninger2,4, Ralf Greiner3, Timothy W. Janzen2, Steven C. Mosimann2, and L. Brent Selinger1

1 Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada
2 Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada
3 Centre for Molecular Biology, Federal Research Centre for Nutrition and Food, D-76131 Karlsruhe, Germany

(RECEIVED December 20, 2006; FINAL REVISION April 2, 2007; ACCEPTED April 3, 2007)

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.

Keywords: inositol polyphosphate phosphatase; protein tyrosine phosphatase; phosphoinositide phosphatase; phytase; myo-inositol; P-loop; hydrolysis pathway


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