Kinetic and structural analysis of a bacterial protein tyrosine phosphatase‐like myo ‐inositol polyphosphatase

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.