Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals， plants， and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution， reaction mechanisms， and regulation. Here， we describe the 2.35-Å-resolution structure of the catalytic core of Siw14， a 5-PP-InsP phosphatase from and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses， thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specifici... More
Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals， plants， and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution， reaction mechanisms， and regulation. Here， we describe the 2.35-Å-resolution structure of the catalytic core of Siw14， a 5-PP-InsP phosphatase from and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses， thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs， but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. () The catalytic P-loop with the CR(S/T) PTP motif contains additional， positively charged residues. () A loop between the α5 and α6 helices， corresponding to the Q-loop in PTPs， contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the α5 helix orientation through intramolecular crowding caused by three bulky， hydrophobic residues. () The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis.
