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 (HCX 5 R) 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 P 6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D-Ins(1,2,4,5,6)P 5 , Ins(2,4,5,6)P 4 , D-Ins(2,4,5)P 3 , and D-Ins(2,4)P 2 . 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 Supplemental material: see www.proteinscience.org Protein tyrosine phosphatase (PTP) superfamily enzymes have been discovered in a range of prokaryotes, and most appear to serve roles that mimic their better-known eukaryotic counterparts as regulators of cellular function (Shi et al. 1998;Kennelly and Potts 1999). Some bacteria have adapted PTPs as ''molecular missiles,'' secreted into the infected host where they assist in the progression of infection (Bliska et al. 1991;Fu and Galan 1998;Bretz et al. 2003). The recently described PTP-like inositol polyphosphate phosphatase (IPPase) from Selenomonas ruminantium, PhyAsr, contains a PTP-like active site signature sequence (HCEAGVGR) but lacks significant primary sequence identity with known IPPases and PTPs (<20%). While its biological function is unclear, it is the first example of a PTP-like enzyme with activity toward myo-inositol hexakisphosphate (Ins P 6 , Fig. 1), one of the most abundant inositol polyphosphates (IPPs) in a majority of prokaryotic and eukaryotic cells (Sasakawa et al. 1995).The X-ray crystallographic structure of PhyAsr (Chu et al. 2004) reveals a PTP-like fold and several novel 4 These authors contributed equally to this work. Reprint requests to: Steven C. Mosimann, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada; e-mail: steven.mosimann@uleth.ca; fax: (403) 329-2057.Article published online ahead of print. Article and publication date are at http://www.proteinscien...

Substrate Binding in Protein-tyrosine Phosphatase-like Inositol Polyphosphatases

“…date, four classes of IPPases have been described that are structurally and mechanistically diverse, including histidine acid phosphatases, ␤-propeller phytases, purple acid phosphatases, and protein-tyrosine phosphatase-like phytases (PTPLPs) (11)(12)(13). Each of the IPPase classes catalyze the stepwise removal of one or more phosphates from IPPs.…”

An insight into protein sequences of PTP-like cysteine phytases

“…All PTPs have a phosphate-binding loop (P-loop) at the base of their active site which contains the characteristic PTP signature sequence C(X) 5 R (Denu and Dixon 1998;Zhang 2003). Site-directed mutagenesis studies have determined that the cysteine residue present in the P-loop is absolutely required for PTP activity (Puhl et al 2007). It is a strong nucleophile, and is easily modified by thiol reagents (Sechi and Chait 1998).…”

“…Biochemical characteristic of CPhy has not been studied in detail and very little literature is available on characterization of CPhy. In one such study, CPhy from Selenomonas ruminantium has been studied and characterized extensively by Puhl et al (2007). In the present study, the 40 reference protein sequences of CPhy from protein databases were retrieved and analyzed 'in silico' for their biochemical features, multiple sequence alignment and identity search, phylogenetic tree construction, distribution of motifs and superfamily using various bioinformatics tools.…”

Current Progresses in Phytase Research: Three‐Dimensional Structure and Protein Engineering

“…The first PTPLP from the anaerobic ruminal bacterium Selenomonas ruminantium (SrPhyA) were characterized [38,39], and demonstrated its protein architecture and catalytic mechanism by crystal structure and mutagenesis studies [40]. Afterward, several sophisticated studies reported the crystal structures of SrPhyA in complex with myo-inositol hexa-, penta-, and tetrakisphosphate to further clarify the substrate-binding mechanism [41][42][43]. The PTPLP proteins fold into a large and a smaller domain (Fig.…”